extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase.

Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis. Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti- PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34cdc2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase. The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34cdc2 kinase.

fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis.
Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti-PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34 ~2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase.
The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34 ~c2 kinase.
OPTOSIS waS originally defined as a program of morphological changes accompanying cell death during which the cells round up and the chromatin condenses, leading to the formation of crescent-shaped masses aggregating at the membrane. In parallel the nucleolus dissolves (Kerr, 1971). These initial changes are followed by cytoplasmic and nuclear compaction, and ultimately by fragmentation of the cell. The nuclear fragments formed in this process are still surrounded by the nuclear membranes (Kerr, 1971;Oberhammer et al., 1993a). As a common biochemical marker for apoptosis, an activation of a nonlysosomal, Ca2+/MgZ÷-dependent endonuclease has been suggested (Wyllie, 1980;Cohen and Duke, 1984). The response is so readily identifiable that endonuclease activation was proposed to be the initiating event in the characteristic condensation of chromatin (Cohen and Duke, 1984). However, it has been shown that incubation of interphase nuclei in extracts prepared from a mitotic cell population induces condensation of the chromatin, dissolution of the nuclear membrane and lamin phosphorylation (Nakagawa et al., 1989;Wood and Earnshaw, 1990). All of these events are most probable due to the activation of p34 ~ kinase and consequent phosphorylation of various nuclear substrates, like the lamins (Gerace and Blobel, 1980;Peter et al., 1990) and histone H1 (Nurse, 1990).
From a morphological point of view, chromatin condensation occurring in an interphase nucleus upon incubation in a mitotic extract looks rather similar to chromatin condensation occurring in an apoptotic nucleus. Furthermore it has been reported that the lamin filament gets phosphorylated during physiological cell death mediated by cytotoxic T-lymphocytes (Ucker et al., 1992).
We therefore decided to compare activation of p34 ~-2 kinase and lamin solubilization during apoptosis and mitosis. A prerequisite was to establish a cell culture system where cells were undergoing apoptosis in a synchronous manner. For this purpose we have used an embryonic fibroblast system. Due to the spindle shape of these cells the processes of rounding up and detachment occur rather slowly as the cells undergo apoptosis at low serum conditions. Thus, these cells stay on the monolayer during the initial process of chromatin condensation. This allows an easy observation of the breakdown of the nuclear lamina by immunohistochemistry and detection of DNA fragmentation by in situ tailing (ISTAIL) ~. Furthermore, these cells can be forced to undergo mitosis after aphidicolin block and thus provides an ideal cell line for comparing apoptosis and mitosis.

Materials
Anti Lamin A+B and p9:agarose beads (p9-beads) were a generous gift from E. Nigg (ISREC, Lausanne, Switzerland), anti-PSTAIR a kind gift from M. Yamashita (National Institute for Basic Biology, O!~Ta~, Japan). Materials for ISTAIL were acquired from Boehringer Mannheim, FRG. Secondary antibodies for the ECL-detection system and reagents were obtained from Amersham, UK.

Cell Culture and Synchronization Experiments
Cell lines, The cell line (HI 1-rasR3) was obtained from primary rat embryo cells transfected with a plasmid coding for human papilloma virus (HPV) type 11 and a plasmid coding for a mutated ras gene. These cells divide rapidly for the first few passages, and then revert to a slow growing phenotype with no HPVI 1 and ras expression being detectable by Northern blots (Cerni et at., 1990). For routine culture, cells were diluted 1:4 (doubling time 36 h) and grown in DMEM medium supplemented with 10% FCS and 200 pg/mi geniticin (G-418 sulphate). In all experiments, epidermal growth factor (EGF, 10 ng/ml) was added.

Cell Culture Experiments
For induction of apoptosis at confluence Hll-rasR3 cells were diluted 1:5 and grown for 4 d to reach 80% confluency. These cells were refed with medium for an additional 40 h before induction of apoptosis at low serum conditions. These were achieved by washing the monolayer twice with unsupplemented DMEM before addition of DMEM supplemented with 0.25% FCS.
For synchronization before S-phase (Figs. 6-8), cells were grown for 4 d to reach 80% confluency. They were refed with medium supplemented with 2 ~g/rnl aphidicolin. After 14 h, cells were released from the aphidicolinblock with two washings with DMEM and directed either into S-G2-M by adding medium supplemented with 10% FCS or into apoptosis by medium supplemented with 0.25% FCS. To follow the onset of DNA synthesis 10 /~M BrdU was added.
For preparation of a mitotic cell population (Fig. 3), monolayers were split 1:15 and grown for 3 d. They were blocked at the entry of S-phase as described above. After 14 h, cells were released and directed into S-G2mitosis by adding medium supplemented with 0.5 /tg/mi nocodazole to arrest cells in mitosis. The monolayers were harvested after an additional 16 h.

Quantification of Apoptotic Nuclei
Glass slides of lO-mm diam were put into 24-well plates or 100-ram dishes before plating. At the indicated time points they were removed and cells 1. Abbreviations used in this paper: AN, apoptotic nuclei; DSB, double strand breaks; E fragment; FIGE, field-inversion gel electrophoresis; HPV, human papilloma virus; ISTAIL, in situ tailing; M, mitotic. fixed with 3 % paraformaldehyde and washed with destilled water before air drying. Samples were stained with 8 ~,g/rnl H33258 for 5 rain, washed with destilled water, and mounted in Mowiol. At each time point two samples were analyzed by counting the number of normal nuclei, apoptotic nuclei, and mitotic figures (M) in 10 microscopic fields (,~1,500 total nuclei). Apoptotic nuclei were further characterized as either with condensed chromatin (AN) before or as nuclear fragments (F) after nuclear fragmentation.

Electron Microscopy
Cells grown on glass slides were forced to undergo apoptosis. Detaching apoptotic cells were separated from ceils in the monolayer and fixed with 2.5% sodium phosphate buffered glutaraldehyde, pH 7.4 and processed as described previously (Oberhammer et at., 1993a).

Immunohistochemistry
For detection of lamins by indirect immunofluorescence (see Fig. 3), cells grown on glass slides were fixed with 3 % paraformaldehyde, washed with destilled water, and air dried. Immunostaining with anti-lamin A+B, or anti-lamin A+C (1:300 dilution) was performed after permeabilization of cells with 0.5% Triton X-100 as described by Naka~awa et al. (1989), followed by incubation with a Texas red-linked anti-mouse Ig (1:500 dilution, Amersham, UK). Samples were counterstained with H33258 (8/~g/mi) before mounting in Mowiol.
For detection of incorporated BrdU (see Fig. 8), cells had to be fixed in 70% ethanol at -20°C. DNA was denatured for 10 rain with 2N HCI at 37°C and the samples neutralized with several washings with 0.1 M HC1, 0.1 M NaBorate, pH 8.5. Anti-BrdU (Boehringer-Mannheim, FRG) was used at a concentration of 1:100, followed by Texas red-linked anti-mouse Ig. The samples were counterstained with H33258 (8 pg/ml) before mounting in Mowiol. For quantitative evaluation, the unlabeled antibody peroxidase-antiperoxidase technique (Steruberger et al., 1970) was used for detection and samples counterstained with hematoxylin.
In $itu Tailing ISTAIL (see Fig. 9) on isolated nuclei was carried out using a modified procedure of the method described by Gold et at. (1993). For rendering the target accessible, we pretreated the fixed samples with a chnotropic agent before digestion with proteinase K. Cells grown on glass slides were fixed 5 rain with 3 % paraformaldehyde, washed with destilled water, and dried at room temperature. Samples were stored at -20°C. Slides were pretreated with Target Unmasking Fluid (TUF-TM, Kreatech Biotechnology, Netherlands). Cells were deproteinated with 20 tzg/mi proteinase K for 20 mill and sequentially washed with PBS and TBS, pH 7.4. At this stage, control samples were treated with 1 ~tg/mi DNAse I in Tris, 10 ~g/nd BSA, pH 7.4 for 20 rain.
Samples were incubated with digoxigenin-DNA labeling mixture and terminal deoxynucleotidyl transferase (0.5 U/~d) in TRIS, 10/~g/mi BSA, 1 mM cobalt chloride, pH 7.4 for 1 h at 37°C. The reaction was stopped with Tris, 20 mM EDTA, pH 7.4 and washed extensively with TBS. An antidigoxigenin alkaline phosphatase labeled (Fab fragments) was used for detection of digoxigenin incorporated into DNA. The alkaline phosphatase developed with Fast Blue Salt until a deep blue stain was obtained (10 rain) in positive controls. The cells were counterstained with HOECHST 33258 before mounting in Mowiol.

Preparation of Samples for Agarose Gel Electrophoresis, Immunoblotting, and Precipitation of p34 ~2 Kinase
Cells were plated onto 100-ram dishes containing two glass slides (10-ram diam) and forced to undergo apoptosis or mitosis. At the indicated time points, the glass slides were taken and the number of normal, apoptotie nuclei and mitotic figures quantified. Cells were scraped from the dish in 5 ml ice-cold PBS supplemented with aportinin, leupeptin, PMSF (5 pg/ml), and 0.05 M NaE In the time course experiments apoptotic cells from the supernatant were harvested by centrifugation and combined with cells in the monolayer. Apoptotic cells were allowed to detach into the superuatant after renewal of the serum free DMEM. After one hour these freshly formed apoptotic cells were collected by centrifugation (A). Cells were pelletted at 1,000 g and suspended in 1 rnl TBS supplemented with aprotinin, leupeptin, PMSF (20 pg/ml), and 0.1 M NaF (ALFNaF).

Gel Electrophoresis
Conventional agarose gel electrophoresis. Cells (2.5 x 106) were collected by centrifugation and suspended in 100/~i of L-buffer (0.01 M Tris-HCI, 0.1 M EDTA, 0.02 M NaCI, pH 7.4) at 37°C and 100/~1 of prewarmed 1% In Cert Agarose in L-buffer (FMC) was added. Four plugs were formed on ice for 10 rain, and then transferred into 5 mi 1% sarcosylate, 20/~g/mi proteinase K in L-buffer and digested overnight at 37°C. The plug and the supernatant were separated. DNA in the supernatant was precipitated with 0.5 vol 7.5 M ammonium acetate and 2 vol of absolute ethanol at -20°C. After centrifugation DNA was dissolved in I00 #1 of 10 mM 1Yis, 10 mM EDTA, pH 8.0. 10 izl was used for conventional gel electrophoresis or deterrnination of DNA content with H33258. After electrophoresis, the gel was stained with ethidium bromide. Gels were incubated with 0.5 ~g RNase-A/mi in running buffer for 2-3 h at 37°C.

Field-inversion Gel Electrophoresis
Field-inversion gel electrophoresis (FIGE) was carried out under the following conditions. Gels were run at 200 V in 0.5* TBE (89 mM Tris, 50 mM boric acid, 2 mM EDTA, pH 8.2) with a pulse rate from TI equal to 2.4 s for the first 3 h and with a ramping rate from T'I equal to 2.4 s to T'2 equal to 84 s for the next 24 h, always with a forward to backward ratio of 3 at 4°C using 1.4% pulsed field certified agaros¢ (Biorad Labs., Hercules, CA). After electrophoresis, the gel was stained with ethidium bromide. Molecular weight markers (Lambda DNA ladder, Saccharomyces cerevisiae chromosomal DNA) were purchased from FMC.

Kinase Assay
Cells (2.5 × 106) were collected by centrifugation and suspended in 1 rul of lysis buffer (50 mM TRIS, pH 7.4, 125 mM NaCI, 1 mM DTT, 0.2% NP-40, 5 mM EDTA, 5 mM EGTA, ALPNaF). After homogenization through a 1-mi syringe equipped with a 25 gauge needle, samples were left on ice for an additional 20 rain. The cytosol was separated from the nuclear pellet by centrifugation at 1,000 g and used for the in vitro kinase assay.
Unspecific adsorption was prevented with the use of HiTrap Protein A columns (Pharmacia LKB Biotechnolngy, Piscataway, NJ) before incubation of 150/zg cytosolic extract protein in 1 ml lysis buffer with 30/~1 of a 50 % suspension of pg-Sepharose beads. After extensive washings with lysis buffer the beads were separated in two portions, one was used to measure histone H1 kinase activity, the other for immunodetection of p34 cdc2 with anti-PSTAIR (as control). The histone H1 kinase assay was performed essentially as described by Peter et al. (1990).

lmmunoblotting
The frozen cells were suspended in 200 t~l RSB (10 mM Tris, pH 7.4, 10 mM NaCI, 2 mM MgCI2, ALPNaF), and 10/Li taken for protein determination. One portion of 100 t~l was treated with 300/zl sample buffer according to Laenunli (1970) before boiling at 95°C for 10 rain (i.e., lysate, L). To the remaining 100/zl, Triton X-100 and 2.5 M NaCI were added to give a final concentration of 1% (vol/vol) and 500 raM, respectively. After 20 min on ice the samples were centrifuged and the pellet resuspended in 200 /~1 RSB. Aliquots (10/~1, corresponding to 8/~g protein) of both pellet (P) and supernatant (S) were analyzed by SDS-PAGE and Western blotting. Membranes were either probed with anti-lamin A+B or first probed with anti-phosphotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY or Biomakor, Israel) followed by reprobing with anti-PSTAIR after removal of the first antibody detection system with 100 mM 2-mercaptcethanoi, 2% SDS, 62.5 mM Tris-HCl, pH 6.7.

Miscellaneous
Protein was determined with the Biorad Assay. DNA content was determined by quantification of the fluorescent H33258-DNA complex in 10 mM

Establishment of a Synchronous System of CeUs Undergoing Apoptosis Using an Embryonic F~broblast upon Transfection with v-ras Together with HPVll
For the purpose of this study it was necessary to have a cell line which undergoes apoptosis in a synchronous manner. In preliminary studies we screened a number of epithelial and fibroblast cell lines for their sensitivity to low serum conditions. These experiments showed that cultures of the Hl l-rasR3 cell line executed an immediate response to low serum conditions which seemed to be more noticeable in areas of high cell density. We therefore prepared confluent monolayers by growing cells until nearly reaching confluence, and then refeeding for an additional 40 h (approximately the duration of one cell cycle) in the presence of EGF. Apoptosis was then induced by low serum conditions and the incidence of cell death determined at different times by phase contrast microscopy and after staining of the same cultures with H33258 (Fig. 1).
Within one hour at low serum conditions, many of the cells showed a change in shape from a flat spindle-like appearance to a rounded one leaving intercellular spaces in the monolayer. This was followed by a more pronounced rounding up (4-6 h), leading finally to detachment of the cells (8-10 h). The detached cells were small with condensed chromatin as shown by phase contrast microscopy and H33258 staining. The morphology of these cells is characteristic of apoptosis.
Electron microscopy (Fig. 2) showed that the nuclear envelope during the whole process of chromatin condensation remained intact. This was true also for the detached apoptotic cells which had started to develop intracellular blebs.

Comparison of Lamin Solubilization and Activation of P34~2 Kinase in Apoptotic and Mitotic Cultures
During mitosis the nuclear lamins are phosphorylated by the activated p34 ~da kinase. This leads to dissolution of the polymer structure by formation of lamin monomers (Gerace and Blobel, 1980;Peter et al., 1990). After dissolution of the nuclear membrane, the monomer dissolves into the cytoplasm and in case of lamin B it has been shown that it is associated with membrane vesicles (Gerace and Blobel, 1980;Stick et al., 1988). We have confirmed these observations in our cell line by indirect immunofluorescence of lamin A+B in mitotic cultures (M) prepared 8-10 h after release from aphidicolin block (Fig. 3). In prophase, the stage where the chromosomes have already condensed and the nuclear membrane has not dissolved yet, lamin A+B condensed in between the chromosomes and at the nuclear periphery. In later stages the epitope was diffusely detected in the cytoplasm.
In apoptotic nuclei (AN), immunofluorescence of lamin A+B was localized around the condensed chromatin. After nuclear fragmentation (F) the epitope seemed to be less and sometimes undetectable. Similar results were observed using anti lamin A+C (data not shown).
Solubilization of lamin A+B was analyzed by centrifugation and separation into soluble (S) and peUetable (P) fractions. The pelletable fraction represents the intact or polymerized lamin and is identified as the 67+70 kD bands on SDS gel electrophoresis (Fig. 4). The soluble fraction contains two identifiable lamin products; the depolymerized  Figure 4. Analysis of solubilization of lamin A+B by West° ern blot and correlation with apoptosis. Cells were forced to undergo apoptosis at low serum conditions. At the indicated times, cells were harvested from both the monolayer and media (i.e., detached cells). Cell lysates were prepared by Triton X-100 + 0.5 M NaCl lysis of the cells and analyzed by SDS gel electrophoresis and Western blotting as described in Materials and Methods. Endpoint controls were prepared by harvesting only the detached apoptotic (A) ceils in the culture media and those ceils arrested in mitosis (M) after nocadaTole inhibition. Solubilization of lamin A+B was determined by immunoprobing with lamin A+B (lamin A+B) oftbe soluble (S) and palletable (P) fraction obtained after centrifugation of the cell lysate. Quantitation of the lamin sub-units was determined by laser densitometric scanning of the films exposed to the blot after application of the ECL-detection system. The latter cells were morphologically mitotic and the predominant lamin form was (as expected) the soluble 67+70 kD form (83 + 6%). The apoptotic cells contained predomi-nantly fragmented nuclei with some condensed chromatin structures and in this case the lamins were mainly found as the degraded 46-kD form (78 ± 10%).
In the time course experiment (Fig. 4) there is a distinct correlation with the morphological changes and the pattern of lamin degradation. Thus, as the number of normal nuclei decreases to 51 ± 4 %, there is a parallel decrease in the intact lamin structure to 64 ± 18%. The increase in apoptotic nuclei is preceded by a small increase in soluble lamins (67+70 kD, maximum 30% at 4 h), and the production of Figure 5. Analysis of p34 ¢dc2 activation. Cells were forced to undergo apoptosis by low serum conditions and harvested as described in Fig. 4 at the indicated times. For comparison, apoptotic cells detaching into the media (.4) and cells arrested at mitosis (M) were analyzed. Tyrosinylation of p34 "de2 kinase was detected in cellular lysates (L) by immunoprobing with anti-p-tyrosine (ptyrosine) followed by reprobing with anti-pstair (pstair). For the histone H1 kinase assay (H/), p34 ¢dc2 was precipitated with p9beads and the precipitated complex used as described in Materials and Methods. Quantitation of phosphorylation of p34 ~d~2 on tyrosine and histone H1 activity was carried out by laser densitometric scanning (H/) of the exposed films. Densitometric analysis of apoptotic (A) or mitotic (M) samples is shown as histograms. Means :t: SEM of four experiments are shown. fragmented nuclei is accompanied by degradation of the larains to the 46-kD fragment. These results demonstrate that the morphological changes in which the nuclei progress from the normal state to condensed chromatin to fragmented nuclei is paralleled by the progressive degradation of the lamin network.
Activation of p34 ~2 kinase before mitosis occurs by a specific dephosphorylation on tyrosine-15 and threonine-14 after association with cyclin B (Nurse, 1990). We investigated the dephosphorylation on tyrosine residues by Western blots. First the membranes were probed with antiphosphotyrosine, followed by reprobing with anti-PSTAIR (Fig. 5). Anti-PSTAIR detects the most conserved amino acid sequence of p34 ~'m. With this method no dephosphorylation of the p34 c~2 kinase was evident after low serum conditions. After normalization of the scan of the 32-kD phospho-tyrosine band to its p34 ~2 counterpart even a slight increase in the phosphorylation of the kinase was detected in apoptotic cells (0 h, 1.0 5= 0.6; M, 0.6 + 0.2; A, 3.2 ± 0.9).
The active p34 ~2 kinase associates not only with the cyclins but also with pl3suc or its vertebrate homologue p9 (Nurse, 1990). We used this protein for precipitation of p34 ~2 and analyzed its histone H1 kinase activity. The kinase showed a slight increase in activity in parallel but not before the increase of apoptosis. However, in apoptotic cells histone HI kinase activity (1.2 + 0.22) was ,vl0-fold lower than that of mitotic cells (9.46 ± 1.55).

Induction of Apoptosis after Release of Cells from Block at S-phase Entry by Aphidicolin
The results shown above suggest that activation of p34 cdc2 kinase apparently does not lead to the chromatin condensation during apoptosis. We furthermore wanted to know if apoptosis and mitosis can be induced in parallel at low serum conditions after release of cells from aphidicolin-block. For comparison, cells were further treated with serum. The incidence of cell death and mitosis was determined by phase contrast microscopy and after staining of the same cultures with H33258 (Figs. 6 and 7). In the first hours at low serum conditions apoptotic cells had a classical appearance of chromatin condensation, whereas later on nuclei with various patterns of chromatin condensation could be observed (Fig.  6). At 4 h at low serum conditions the incidence of apoptotic nuclei rose to 12.8 -t-1.7% (Fig. 7) and stayed at this percentage thereafter (14.7 + 0.3% 10 h at low serum conditions). Both in serum-treated and serum-deprived cultures, comparable numbers of cells started to undergo mitosis 6 h after release from the aphidicolin block (Figs. 6 and 7). This led to the paradox that 10 h after release of the S-phase block the rate of mitosis was slightly higher in the apoptotic cultures (3.7 + 0.9) than in the mitotic ones (3.0 ± 1.3%).
For detection of cells in S-phase we looked at the incorporation of BrdU after release from aphidicolin-block with an immunohistochemical technique (Fig. 8). We noticed that at low serum conditions cells were able to replicate DNA. Apoptotic cells positive for BrdU could be observed. This indicates that these cells synthesized DNA before they received the signal to undergo apoptosis.

Analysis of DNA Fragmentation
We wanted to compare the appearance of DNA double strand breaks (DSB) and further digestion of chromatin with the stages of chromatin condensation. We analyzed DNA strand breaks by ISTAIL (Fig. 9) and pulse field gel electrophoresis followed by conventional gel electrophoresis (Fig. 10). In preliminary experiments we found that both the cellular and chromatin condensation prevented the access of enzymes and antibodies to their targets (Fig. 9 a). However, pretreatment of cells with a chaotropic agent followed by removal of proteins by proteinase K digestion rendered the condensed chromatin accessible to ISTAIL and antibody labeling (Fig. 9 b). With this method no background digoxigenin labeling was detectable in normal nuclei. DSB were detectable during the initial stages of chromatin condensation. The digestion starts around the areas of condensed chromatin (Fig. 9 c). The complete genomic digestion takes hours and apoptotic cells detaching into the supernatant contain partly digested DNA (data not shown). Fig. 10 shows the appearance of DNA fragments detected by gel electrophoresis. As expected from our experiments (Oberhanuner et al., 1993b) the fragmentation of DNA into domain-sized fragments parallels the morphological onset of Oberhammer ¢t al. Degradation of Nuclear Lamins Accompanies Apoptosis Figure 6. Morphology of cells directed into mitosis or apoptosis after release from S-phase block. After release from the aphidicolin-block (0 h) cells were either directed into S-G2-M by adding medium supplemented with 10% FCS (+FCS) or into apoptosis by medium supplemented with 0.25% FCS only (-FCS). The cells were observed by phase-contrast microscopy (phase contrast) before stainin~ the DNA with H33258 (H33258). For a better demonstration of the chromatin condensation, H33258 stained nuclei are shown at a higher magnification. Bars, l0 tLm. apoptosis (Fig. 10). The 50-kbp DNA is progressively degraded to smaller sizes as revealed by conventional gel electrophoresis of the short chain DNA migrating out of the plug during digestion with proteinase K. Quantification of this DNA did not show a linear correlation with the amount of apoptotic cells in the culture. In detached apoptotic cells most of the DNA consists of 300 and 50 kbp (Fig. 10) and only 14 ± 2% of the DNA is <50 kbp.

D i s c u s s i o n
It is well established that most of the mitotic processes are triggered by tyrosine dephosphorylation of the p34 ~2 corn-plex leading to an increase in kinase activity enabling the complex to phosphorylate various substrates, like e.g., lamins or histone HI (for reviews see Draetta, 1990;Nurse, 1990;Murray and Kirschner, 1989). Furthermore it has been shown that extracts containing activated p34 '~2 kinase prepared from Xenopus eggs (Newport and Spann, 1987;Adachi et ai., 1991), clam oocytes (Dessev et al., 1991) or cells synchronized in mitosis (Nakagawa et al., 1989;Wood and Earnshaw, 1990) activate the processes of chromatin condensation and lamin solubilization in isolated nuclei. Therefore we hypothesized that some of the processes responsible for mitotic chromatin condensation might also be activated during apoptosis. For this study, we established After release of cells from the aphidicolin block and further treatment with serum ( ) or without serum ( ..... ), normal nuclei (NN), apoptotic nuclei (AN+F), and mitotic figures (M) were a highly synchronized system of H11-rasR3 cells undergoing apoptosis and examined the changes in lamin structure. The results can be summarized as follows: (1) There is a small increase in lamin solubilization before the onset of apoptotic chromatin condensation. (2) l.amin degradation parallels chromatin condensation. (3) In apoptotic cells the tyrosine residues of p34 ~2 are not dephosphorylated. (4) In apoptotic cells p34 ~c2 kinase is nearly 10-fold less active than in mitotic cells. (5) Apoptosis is more easily induced in cells in S-phase than in M-phase. (6) DNA strand breaks, assayed by ISTAIL, were detected in initial stages of chromatin condensation around the areas of condensed ehromatin. (7) The appearance of high molecular weight DNA of multimers of 50-kbp bands, as detected by FIGE, parallels chromatin condensation. (8) Apoptotic cells contain mainly digested lamin A+B with the DNA degraded mainly into large fragments (300-350 kbp) with only a small amount <50 kbp (14%). From these findings we can make the following conclusions and these are discussed below.

Chromatin Condensation during Apoptosis Occurs without a Parallel Increase in p34~2 Kinase
The data shown in this paper show that p34 ~-: is only slightly activated during apoptosis and that apoptosis could be far more induced in parallel with S-phase than in parallel with M-phase. It has been shown that chromatin condensation and fragmentation of DNA into oligosomes characteristic of apoptosis can be induced in isolated nuclei by incubation in an extract prepared from cells after a sequential S-phase/M-phase synchronization (Lazebnik et al., 1993). Upon depletion of activated p34 ~2 kinase by p9 the extract was still able to produce chrornatin condensation and fragmentation of DNA. Furthermore, chromatin condensation and DNA fragmentation could not be observed if the extract was prepared from M-phase cells not presynchronized in S-phase with aphidicolin. This correlates well with our observations. evaluated in H33258 stained cultures. Means + SEM of four experiments are shown. Figure 8. Incorporation of BrdU in cells directed into mitosis or apoptosis after release from S-phase block. After 14 h, cells were released from the aphidieolin-block with two washings with DMEM and directed either into S-G2-M by adding medium supplemented with 10% FCS (a and b) or into apotosis by medium supplemented with 0.25% FCS (c-h). To follow the onset of DNA synthesis 10 #M BrdU was added. At different timepoints, cells were fixed with 70 % ethanol and further processed for indirect immunofluorescence of BrdU. Staining with H33258 (a, c, e, and g) and with corresponding Texas-red fluorescence indicating brdU (b, d, f, and h) is shown for the timepoints which are 2 (a-d), 6 (e and f), or l0 h (g and h) after treatment. Bar, 10 #m. All samples were fixed with 3 % paraformaldehyde 5 h after low serum conditions. Positive controls (a and b) were normal nuclei treated with DNase I. In a the pretreatment with the chaotropic agent which leads to swelling of the chromatin was omitted. The arrow in c indicates first stage of chromatin condensation in an apoptotic nucleus. Bar, 10 tLm.

Nuclear Lamins Are Degraded in Parallel with Chromatin Condensation
Using anti lamin A + B we observed that the filamentous structure of the lamin network disappears during apoptosis, due to a time-dependent degradation. Digestion of nuclear lamin has also been found in physiological cell death mediated by cytotoxic T-lymphocytes (Licker et al., 1992) and apoptosis induced by etoposide (Kaufmann, 1989).
The identity of the enzyme(s) responsible for lamin degradation is unknown. However in the nuclear scaffold a Ca ~+ dependent protease exists which shows a marked selectivity for lamins (TOk6s and Clawson, 1989). The localization and activity of lamin degradation (,070% of the lamins are destroyed within 60 min) of this enzyme would make it a good candidate for digestion of nuclear proteins during apoptosis. Apoptotic cells were prepared as described in Fig. 4. Before analysis at the indicated times, cells were harvested from both the monolayer and the media. As in Fig. 4, for comparative purposes, apoptotic cells (A) detaching into the supernatant were analyzed. The percentage of apoptotic cells (%A) indicates the percentage of apoptotic nuclei quantified in cells grown on glass 10-mm glass cover slides which were included in the normal culture dishes. Cells were embedded in agarose plugs and after digestion with proteinase K plugs were used for FIGE (upper picture). Short chain DNA having migrated out of the plug were quantified by the fluorescent H33258-DNA complex (DNA) analyzed by conventional gel electropboresis (lower picture). As markers the l.ambda DNA ladder (L)~and Saccharomyces cerevisiae DNA standards (S) were used.

Is DNA Fragmentation also a Result of the Proteolysls of Nuclear Proteins?
In the case of apoptosis mediated by etoposide it has been shown that there is a cleavage of the poly(ADP-ribose) Polymerase (Kaufmann et al., 1993) and digestion of other nuclear proteins like topoisomerase I and II, histone 1-11 and p67 (Kaufmann, 1989). Protease digestion of topoisomerase II removes its COOH-terminal end revealing a core enzyme with full decatenation activity (Shiozaki and Yanagida, 1991). Thus proteolysis oftopoisomerase 1I during apoptosis could activate the protein. Topoisomerase 1I (Adachi et al., 1989;Earnshaw et al., 1985;Filipski et al., 1990;Gasser and Laemmli, 1987) could be responsible for the formation of the large domaine-sized (50 kbp) fragments of the DNA during apoptosis. In all cell lines studied so far, the appearance of these high molecular mass DNA bands detectable by FIGE (Walker et al., 1991;Brown et al., 1993;Oberhammer et al., 1993b;Tomei et al., 1993) parallels chromatin condensation, typical of apoptosis. It has been shown that incubation of interphase nuclei in mitotic extracts leads to different condensation patterns depending on the amount of Topoisomerase II present in a nucleus (Wood and Earnshaw, 1990;Adachi et al., 1991). In the Xenopus reconstitutive system, a nearly linear correlation between the degree of chromatin condensation and the amount of topoisomerase II was observed. Topoisomerases themselves are able to nick DNA around their specific DNA attachment sites (SAR regions) in the flanking regions (circe effect, Mirkovitch et al., 1984;Adachi et al., 1989). It has to be emphasized that in an apoptotic nucleus the amount of short chain DNA (450 kbp) represents only a small amount of the total DNA. Thus a transient activation of topoisomerase II before its complete degradation could also account for the appearance of oligosomal fragments detectable by conventional gel electrophoresis.


extfieldsofstudy: Biology Medicine

text:
Synaptic activity and connective tissue remodeling in denervated frog muscle

Denervation of skeletal muscle results in dramatic remodeling of the cellular and molecular composition of the muscle connective tissue. This remodeling is concentrated in muscle near neuromuscular junctions and involves the accumulation of interstitial cells and several extracellular matrix molecules. Given the role of extracellular matrix in neurite outgrowth and synaptogenesis, we predict that this remodeling of the junctional connective tissue directly influences the regeneration of the neuromuscular junction. As one step toward understanding the role of this denervation-induced remodeling in synapse formation, we have begun to look for the signals that are involved in initiating the junctional accumulations of interstitial cells and matrix molecules. Here, the role of muscle inactivity as a signal was examined. The distributions of interstitial cells, fibronectin, and tenascin were determined in muscles inactivated by presynaptic blockade of muscle activity with tetrodotoxin. We found that blockade of muscle activity for up to 4 wk produced neither the junctional accumulation of interstitial cells nor the junctional concentrations of tenascin and fibronectin normally present in denervated frog muscle. In contrast, the muscle inactivity induced the extrajunctional appearance of two synapse-specific molecules, the acetylcholine receptor and a muscle fiber antigen, mAb 3B6. These results demonstrate that the remodeling of the junctional connective tissue in response to nerve injury is a unique response of muscle to denervation in that it is initiated by a mechanism that is independent of muscle activity. Thus connective tissue remodeling in denervated skeletal muscle may be induced by signals released from or associated with the nerve other than the evoked release of neurotransmitter.

Abstract. Denervation of skeletal muscle results in dramatic remodeling of the cellular and molecular composition of the muscle connective tissue. This remodeling is concentrated in muscle near neuromuscular junctions and involves the accumulation of interstitial cells and several extracellular matrix molecules. Given the role of extracellular matrix in neurite outgrowth and synaptogenesis, we predict that this remodeling of the junctional connective tissue directly influences the regeneration of the neuromuscular junction. As one step toward understanding the role of this denervation-induced remodeling in synapse formation, we have begun to look for the signals that are involved in initiating the junctional accumulations of interstitial cells and matrix molecules. Here, the role of muscle inactivity as a signal was examined. The distributions of interstitial cells, fibronectin, and tenascin were determined in muscles inactivated by presynaptic block-ade of muscle activity with tetrodotoxin. We found that blockade of muscle activity for up to 4 wk produced neither the junctional accumulation of interstitial cells nor the junctional concentrations of tenascin and fibronectin normally present in denervated frog muscle. In contrast, the muscle inactivity induced the extrajunctional appearance of two synapse-specific molecules, the acetylcholine receptor and a muscle fiber antigen, mAb 3B6. These results demonstrate that the remodeling of the junctional connective tissue in response to nerve injury is a unique response of muscle to denervation in that it is initiated by a mechanism that is independent of muscle activity. Thus connective tissue remodeling in denervated skeletal muscle may be induced by signals released from or associated with the nerve other than the evoked release of neurotransmitter.
ENERVATION of skeletal muscle results in striking changes in the cellular and molecular composition of the muscle connective tissue. Interstitial cells accumulate selectively in junctional regions of skeletal muscles, near neuromuscular junctions, after damage to motor axons (6,16,45). Experimental evidence suggests that these interstitial cells are fibroblasts, cells that produce extracellular matrix (6). The redistribution of several extracellular matrix molecules is coincident with the accumulation of interstitial cells; fibronectin, tenascin, and heparan sulfate proteoglycan become concentrated in junctional regions of denervated muscle (16,46). The hypothesis that connective tissue remodeling in junctional regions of denervated muscle may influence the reinnervation of muscle is suggested by evidence that several extracellular matrix molecules are involved in aspects of synaptogenesis (for review see reference 44). Neurite outgrowth and elongation in vitro is promoted by laminin and fibronectin (26,30,42). In vivo, neurons regenerate axons along the laminin-rich Schwann cell basal lamina sheaths to reinnervate muscle fibers (21,24). The assembly of postsynaptic specializations, clusters of acetylcholine receptors (AChR) l and acetylcholinesterase, is initiated in vivo by molecules in the muscle fiber basal lamina (1,31) and in vitro by the matrix molecule, agrin (15,35,39,47,48).
One step toward determining the role of the junctional interstitial cells in the formation of neuromuscular junctions is the identification of the signals that initiate the connective tissue remodeling following denervation. The level of muscle activity is one variable altered by denervation and is known to influence the structure and molecular organization of the muscle. For example, in normal innervated muscle, muscle fibers are sensitive to acetylcholine (ACh) at neuromuscular junctions; AChRs are concentrated there (25). In contrast, muscle fibers made inactive by either denervation or blockade of synaptic transmission display dramatically increased sensitivity to ACh in extrajunctional muscle regions due to insertion of AChRs throughout the muscle fiber membrane (3,13,28,29,32). This development of extrajunctional sensitivity to ACh in inactive mammalian muscles can be in-hibited or reversed by restoration of muscle activity with electrical stimulation (12,23,29).
The experiments presented here address the role of muscle activity in initiating the remodeling of the junctional connective tissue in denervated frog muscle. Muscles were made inactive by presynaptic blockade of synaptic activity with tetrodotoxin (TTX), a poison that blocks voltage-sensitive sodium channels (33). This pharmacological blockade of muscle activity is referred to as functional denervation. We have determined that denervation-like remodeling of the junctional connective tissue does not occur in functionally denervated muscles. There was no junctional accumulation of interstitial cells; the interstitial cell densities of functionally denervated muscles were indistinguishable from those of normal innervated muscles. Further, consistent with the hypothesis that the accumulated interstitial cells are responsible for matrix remodeling after denervation, denervationlike accumulations of the extracellular matrix molecules tenascin and fibronectin did not appear in inactive muscles. In contrast, muscle inactivity did induce known denervation responses in frog muscle fibers; sensitivity to ACh and the synaptic muscle antigen mAb 3B6 were detected in extrajunctional regions of functionally denervated muscle fibers. These data demonstrate for the first time an activity dependence of denervation responses of frog muscle and suggest that muscle inactivity does not initiate the junctional connective tissue remodeling in denervated muscle. Thus, unlike the activity-dependent responses of denervated muscle fibers, the connective tissue response to denervation is mediated by a mechanism that is independent of muscle activity.

Muscles
Each muscle fiber of the paired cutaneous pectoris muscle of the frog, Rana pipiens, is singly innervated in its center third by the cutaneous pectoris nerve, resulting in clearly defined junctional and extrajunctional regions (6). Due to the arrangement of muscle fibers, the anterior tibialis muscle has neuromuscular junctions all along its length so that each cross section contains a group of muscle fibers sectioned through the junctional region.

Denervation
The innervation to the cutaneous pectoris was severed bilaterally either at the level of the forelimb and entrance of the nerve into the muscle or, for longer periods of denervation, as the brachial nerve exited the vertebral column (27). The anterior tibialis muscle was denervated unilaterally by severing the seventh, eighth and ninth spinal nerves proximal to their fusion to form the sciatic nerve.

General Procedures for Light Microscopy
Cutaneous pectoris muscles were dissected in Ringer's solution (115 mM NaCI, 2 mM KCI, 1.8 mM CaCI2, 1 mM NaH2PO4H20, pH 7.2), fixed for 30 min in 0.8% glutaraldehyde in 0.09 M phosphate buffer, stained for cholinesterase, refixed for 1 h in 1% osmium tetroxide in 0.09 M phosphate buffer, dehydrated in ethanol, and embedded flat in a mixture of LX-112 and araldite (6). 1 /zm cross sections of junctional and extrajunctional muscle regions were stained with toluidine blue.

Presynaptic Blockade of Muscle Activity
Cutaneous pectoris nerve activity was blocked for 2 wk by implantation of a unidirectional 1.5 mm time-release pellet containing 9 p.g TTX (Innovative Research of America, Toledo, OH). The 9 ~g dose of TTX was chosen since nerve activity was restored prematurely at a lower dose (4.5/zg TTX) and the activity blockade with a higher dose (13.5/~g TTX) resulted in decreased survival and was so extensive as to prevent successful stimulation of the nerve distal to the pellet site. Frogs were anesthetized by immersion in 0.15 % tricaine manthanesulfonate in water (pH 7.0) and a TTX or placebo pellet was placed on the cutaneous pectoris nerve at least 1 mm from its entrance to the muscle. The skin incision was closed with suture. Nerve activity was blocked at the earliest time at which the blockade of activity was assessed, 48 h after pellet implantation. For longer periods of activity blockade (3-4 wk), the pellet was removed and replaced after 2 wk. Frogs fully recovered from pellet implantation within 24 h and resumed normal levels of activity.
The effectiveness of the blockade of synaptic transmission by TTX and the integrity of the cutaneous pectoris nerve were assessed at the time of dissection both in situ and after the muscle was removed. To determine if synaptic transmission was blocked, the nerve was electrically stimulated (1 Hz, 10 ms duration, 0.1-20 V) first proximal and then distal to the pellet and the cutaneous pectoris was observed for evidence of contractions. TTXtreated nerves were challenged with voltage pulses 3-10 times greater than those needed in control preparations to elicit contractions (1-2 V). Muscles were included in the analysis only if muscle contractions were elicited by stimulation of the nerve distal but not proximal to the TTX pellet. Further, muscles for interstitial cell density determination and electrophysiological recording were fixed, embedded in a plastic wafer as described above, and examined in whole mount for the presence of degenerating axons. Disruption of the myelin sheath was considered an indicator of axonal damage when assessing the integrity of individual axons (Fig. 1). Innervated (control, TTX, and placebo-treated) preparations that displayed damage to more than one axon were excluded from the analysis.

Determination of Interstitial Cell Density
Interstitial cell density was quantitated in junctional and extrajunctional sections of each muscle as previously described (6). Briefly, the number of interstitial cell nuclei/muscle fiber was determined for at least five fields in each section and a mean density was obtained. For junctional sections, a field contained several muscle fibers with cholinesterase-stained synaptic sites, Cell density was determined without knowledge of the type of preparation (control, TTX, and placebo-treated).

Length of Neuromuscular Junctions
Cutaneous pectoris muscles were fixed, stained for cholinesterase, and embedded flat as described above. The linear extent of individual neuromuscular junctions, marked by cholinesterase stain, was measured with an eyepiece micrometer at 200x.

Immunohistochemistry
Cutaneous pectoris muscles were dissected in sylgard-coated petri dishes and the borders of junctional regions were marked with pins. To reduce the damage due to thawing during mounting, muscles were sandwiched between other frog muscles and were frozen in liquid nitrogen. Junctional regions of muscles were cut out and mounted with Tissue-Tek O.CX. compound (Miles Inc., Elkhart, IN). Anterior tibialis muscles were dissected, frozen, and a block of muscle was mounted in a similar fashion. Ten micrometer cross sections were collected on gelatin-coated slides and stored at -20"C. Sections were fixed for 5 min in I% formalin in PBS (137 mM NaCl, 1.5 mM KH2PO4, 2.7 mM KCI, 8.0 mM Na2HPO4), washed for 5 min in 0.1% Triton-X 100 in PBS (PBST), incubated for l0 rain in blocking solution (3% bovine serum albumin, 0.2% Tween 20, 5% goat serum in PBS), and incubated for 1 h at room temperature in primary antibody. Sections were then washed for 30 min in PBST, incubated for 1 h in fluorochrome-labeled secondary antibodies, and washed for 30 rain in PBST. Primary and secondary antibodies were diluted in blocking solution prior to incubation. Rhodamine-labeled c~-bungarotoxin (Molecular Probes, Eugene, OR) was added to the secondary incubation to mark neuromuscular junctions. Coverslips were mounted with Citifluor mountant medium AFI (City University, London).

Antibodies
The primary antibodies directed against the extracellular matrix molecule fibronectin were a rabbit polyclonal directed against Xenopus fibronectin In addition, mAb 3B6, a mouse IgM that stains an intracellular muscle epitope at the frog neuromuscular junction was used (7). Secondary antibodies were labeled with FITC and included goat anti-rabbit IgG, goat anti-mouse IgM, and afffinity-purified goat anti-mouse IgG (Cappel Research Products, Durham, NC).

lontophoresis of Acetylcholine
Cutaneous pectoris muscles were dissected in a Ringer's solution (110 mM NaCI, 2 mM KCI, 0.5 mM CaCI2, 5 mM MgCI2, pH 7.4). Determination of ACh sensitivity was similar to the method of del Castillo and Katz (11). Muscle fibers in the lateral portion of the muscle were impaled about 1 mm from the main nerve trunk; there neuromuscular junctions were consistently found. The junctional location of the recording electrode was confirmed in innervated and activity-blocked muscles by the detection of miniature endplate potentials (MEPPs). In general, membrane potentials of muscle fibers included in the analysis ranged from -8 0 to -9 0 mV though recordings were made at -6 0 mV in a few functionally denervated fibers. ACh pulses were delivered at the site of the recording electrode by iontophoretic micropipets (3 M ACh chloride; Sigma Chemical Co., St. Louis, MO). ACh release currents were normally limited to 100 nA with a 10 ms duration and produced ACh potentials 1-5 mV in amplitude. Longer pulse durations (up to 50 ms) were used in extrajunctional regions of some functionally denervated muscle fibers. After the first impalement and application of ACh, electrodes were moved a measured distance (50-300 mm) along the muscle fiber where the same fiber was reimpaled and again pulsed with ACh. This was repeated along the muscle fiber until no response was recorded. In some instances, impalement and iontophoresis were in clear extrajunctional muscle regions; the impalement sites were greater than 4 mm from the entrance of the nerve into the muscle and at least 2 mm from the insertion of the fibers into the skin. In addition, neither axons nor cholinesterase stain were detected near these extrajunctional recording sites. Voltage responses of muscle fibers were recorded on a storage oscilloscope and photographed. ACh sensitivity was computed as the ratio of amplitude of the ACh potential (mV)/charge of ACh current (nC) (32). Data are presented as mean + SD.

Muscle Activity and Interstitial Cell Density
A first set of experiments determined that pellet implantation itself did not induce or block the junctional accumulation of interstitial cells. The interstitial cell density in placebotreated innervated preparations was not different from that Figure 2. The distribution of interstitial cell nuclei in innervated and denervated muscle is not altered by a 2 wk implantation of placebo pellets. The number of muscles in each group (n) are innervated (11), innervated + placebo (6), 2 wk denervated (8), and 2 wk denervated + placebo (8). Data are shown as the mean ± SD.
in normal muscles (Fig. 2). Further, after 2 wk of denervation, equivalent junctional accumulations of interstitial cells resulted in muscles with or without placebo pellets.
Whereas true denervation resulted in a striking accumulation of interstitial cell nuclei in junctional regions of denervated muscles as previously described (6), we found no junctional accumulation of interstitial cells in muscles functionally denervated for 2-4 wk (Figs. 3 and 4). Instead, the connective tissue morphology of junctional regions of functionally denervated muscles closely resembled that of control innervated muscles; there were few interstitial cell nuclei in the endomysium and perimysium of either type of muscles. This distribution of interstitial cells was maintained in muscles functionally denervated for up to 4 wk, indicating that the onset of the interstitial cell accumulation was not simply delayed in functionally denervated preparations. Further, overall interstitial cell density was unaltered by muscle inactivity; the density of interstitial cells in extrajunctional muscle regions of 4 wk functionally denervated muscles (0.16 + 0.07 SD, n = 5 muscles) was not different from control innervated values (0.15 + 0.06, n = 18). The failure of interstitial cells to accumulate in functionally denervated muscles was not due to a direct effect of TTX since the interstitial cell density was not different in control muscles after 2 wk of denervation (0.50 + 0.19 SD, n = 7 muscles) when compared to muscles after 2 wk of denervation and TTX treatment (0.66 + 0.15, n = 6). These data demonstrate that the junctional accumulation of interstitial cells in denervated frog muscle is not regulated by muscle activity. Thus, denervation must provide other signals that induce the interstitial cells to proliferate and accumulate near denervated synaptic sites (6).

Muscle Activity and Distribution of Matrix Molecules
Following denervation of rat skeletal muscle, the muscle extracellular matrix is remodeled in that fibronectin, tenascin and heparan sulfate proteoglycan become concentrated in junctional regions (46). Evidence that interstitial cells isolated from denervated rat muscle produce these matrix molecules suggests that the interstitial cells are responsible for the junctional matrix remodeling in denervated muscle (16). We have found a similar junctional accumulation of the matrix   . The distributions of tenascin and fibronectin immunoreactivity are strikingly altered by denervation; both tenascin and fibronectin are highly concentrated in the junctional connective tissue of 2 Wk denervated muscles (Fig. 5). To determine the role of muscle activity in this remodeling of the muscle extracellular matrix, we examined the distribution of tenascin and fibronectin in muscles functionally denervated for up to 4 wk. The distribution of the matrix molecules tenascin and fibronectin was not altered by functional denervation. In four muscles (four frogs) functionally denervated for 4 wk, denervation-like junctional accumulations of tenascin stain were not observed (Fig. 6). Similarly, there was no such redistribution of tenascin after either 2 or 3 wk of muscle inactivation (data not shown). The pattern Of tenascin stain in functionally denervated muscles was not however identical to that in control innervated muscles. In 4 wk functionally denervated muscles, structures in the muscle interstitial space, presumably nerve bundles, occasionally displayed tenascin immunoreactivity and the staining of synaptic sites was more prevalent. This tenascin immunoreactivity was very limited and not equivalent to the induction of tenascin by denervation. The pattern of fibronectin stain in 2-4 wk functionally denervated muscles resembled controls; there was limited fibronectin stain in both junctional and extrajunctional regions (Fig. 6). These results indicate that the interstitial accumulations of the matrix molecules tenascin and fibronectin in junctional regions after denervation are not regulated by the level of muscle activity. These results are consistent with the hypothesis that the interstitial cells are responsible for the matrix remodeling observed after denervation. The failure of blockade of synaptic activity to induce remodeling of the muscle connective tissue may be due to either ineffective blockade of synaptic transmission or a failure of frog muscle to display activity-dependent denervation responses. Previous evidence of induction of denervation responses by muscle inactivity comes from mammalian preparations (3,4,9,12,14,18,23,29,37,41,50). The effectiveness of the TTX blockade of muscle activity was tested by electrical stimulation of the cutaneous pectoris nerve both proximal and distal to the TTX pellet. Only muscle preparations that contracted with distal nerve stimulation and not proximal stimulation were included in the analysis. As further evidence of the effectiveness of the TTX blockade and the activity dependence of frog muscle denervation responses, we examined the distribution of two synaptic molecules in functionally denervated muscles.

Muscle Activity and Acetylcholine Sensitivity
We first determined the distribution of AChR in functionally denervated muscle fibers by assaying ACh sensitivity. In mammalian muscle, extrajunctional ACh sensitivity is one of several denervation responses known to be induced by blockade of muscle activity (12,23,29). Though extrajunctional ACh sensitivity has been demonstrated in denervated amphibian muscle (13,32), its activity dependence has not been documented. We used iontophoretic application of ACh to map the ACh sensitive membrane on individual muscle fibers from innervated, denervated, and functionally denervated muscles. a rising phase of 100-300 ms (Fig. 7 a). Occasionally no ACh potentials were detected though MEPPs were present and it was assumed that the neuromuscular junction was on the opposite surface of the muscle fiber. In normal innervated muscle fibers, ACh potentials (maximal sensitivity: 9.6 + 3.2 mV/nC, n = 7 muscle fibers) were detected for an average distance of 0.4 mm (+ 0.17, n = 7) from the original impalement site (Fig. 8 a). The measured lengths of AChsensitive membrane were consistent with neuromuscular junction lengths determined by cholinesterase stain; neuromuscular junctions ranged from 0.2-1.2 mm in length with an average length of 0.6 mm (+ 0.19 SD, n = 150 muscle fibers). Denervated Muscle. 2 wk after denervation, ACh potentials were detected at greater distances along muscle fibers, up to 5 mm from the original impalement site. Though the absence of MEPPs prevented the direct determination of the position of neuromuscular junctions, original impalement sites in denervated muscle were likely at endplates since the recordings were made in the same endplate-rich areas in both denervated and innervated muscles. In addition, the maximal ACh sensitivity at denervated junctional regions (12.7 + 5.3 mV/nC, n = 5 muscle fibers) was similar to that of innervated muscles. Unlike innervated muscle fibers where ACh sensitivity dropped off less than a millimeter from the impalement site, ACh sensitivity remained elevated in extrajunctional regions of denervated muscle fibers (Fig. 7 b). Extrajunctional ACh sensitivity was about 1/10 of the sensitivity at the neuromuscular junction (Fig. 8 b), a finding consistent with previous reports (13,32).
Functionally Denervated Muscle. After 2 wk of blockade of synaptic activity by TTX, the distribution of ACh sensitivity was strikingly similar to that of normal innervated muscles (Fig. 7 c), The presence of MEPPs at neuromuscular junctions indicated that the nerve was intact. ACh sensitivity at endplates (10.2 + 3.0 mV/nC, n = 6 muscle fibers) was similar to that of innervated and 2 wk denervated muscle fibers. The ACh sensitivity remained localized to the neuromuscular junction; ACh potentials were detected on average 0.51 nun (5= 0.12, n = 6 muscle fibers) from the first impalement site (Fig. 8 c). In one 2 wk TTX preparation, extrajunctional sensitivity to ACh was detected but the lack of MEPPs and inspection of the muscle after fixation revealed that the nerve had degenerated and the muscle was denervated. Distance from Original Impalement (mm) When the period of muscle activity blockade was extended to 3 wk, sensitivity to ACh was detected in extrajunctional muscle regions (Fig. 9). In four muscle fibers from two frogs ACh potentials were recorded several millimeters from cholinesterase-stained neuromuscular junctions. There was no evidence of axon damage in these preparations. ACh pulses of similar size and duration failed to induce ACh potentials in the same extrajunctional positions in a normal innervated muscle. The extrajunctional ACh sensitivity of muscle fibers after 3 wk of functional inactivity (3.8 + 2.3, n = 3 muscle fibers, two frogs) was similar to that of muscle fibers denervated for 2 wk (Fig. 8). In two other functionally denervated muscle fibers, ACh potentials were produced only in response to very long ACh pulses (90-150 ms); these were not considered positive responses.

Muscle Activity and Distribution of a Muscle Fiber Antigen
In normal muscle fibers, the muscle antigen recognized by mAb 3B6 is concentrated at the neuromuscular junction (7). After denervation, the mAb 3B6 antigen appears in extrajunctional regions of the muscle fiber perimeter as well as the cytoplasm. The distribution o f m A b 3B6 staining was examined in muscles functionally denervated with TTX (Fig.  10). After 2 wk of activity blockade, mAb 3B6 stain remained concentrated at neuromuscular junctions of most muscle fibers with only limited nonsynaptic stain on a few muscle fibers (data not shown). After 3--4 wk of activity blockade, a denervated pattern of mAb 3B6 stain was de- Figure 10. The synaptic muscle antigen mAb 3B6 appears in nonsynaptic regions of the muscle fibers after loss of muscle activity. The junctional distribution of mAb 3B6 is shown for an (a) innervated, (b) 4 wk denervated, and (c) 4 wk functionally denervated muscle.
Rhodamine-labeled ot-bungarotoxin (d-f) marks the location of neuromuscular junctions. Bar, 40/xm. tected; there was a significant level of mAb 3B6 staining in extrajunctional regions of muscle fibers (Fig. 10).
These data demonstrate that the presynaptic blockade of synaptic activity was effective and sufficient to induce known denervation responses of muscle fibers, extrajunctional appearance of ACh sensitivity and the mAb 3B6 antigen. This is the first demonstration that a denervation response of frog muscle can be induced by blockade of muscle activity. Further, these results suggest that denervation responses of frog muscle fibers are not initiated solely by muscle inactivity since the development of extrajunctional ACh sensitivity and mAb 3B6 immunoreactivity was slowed in muscles made inactive by TTX when compared to denervated muscles.

Discussion
The experiments presented here examined whether loss of muscle activity was a sufficient signal to generate the cellular and molecular connective tissue remodeling normally observed in junctional regions of denervated frog skeletal muscle. The activity of innervated muscles was modified by presynaptic blockade of synaptic transmission with TTX. In muscles functionally denervated for up to 4 wk no denervation-like accumulations of either interstitial cells or the matrix molecules tenascin and fibronectin were detected. In contrast, similar periods of inactivity were sufficient to induce the redistribution of two synaptic molecules, AChR and the mAb 3B6 antigen. These results indicate that remodeling of the junctional connective tissue in response to denervation is initiated by a mechanism independent of muscle activity.
We first established the effectiveness of the blockade of muscle activity by TTX. For inclusion in our analysis, a muscle inactivated by TTX had to meet two criteria. First, muscle had to be functionally denervated at the time of dissection. This was determined by stimulation of cutaneous pectoris nerve proximal to the location of the TTX pellet. In all but two preparations treated with 9 #g of TTX, the muscles failed to contract in response to stimulation of the nerve. Second, the cutaneous pectoris nerve had to be intact and undamaged by the pellet. This was assessed at the time of dissection by evoking muscle contractions in response to nerve stimulation distal to the TTX pellet. Since it was not possible to determine that all muscle fbers responded to stimulation, the nerve integrity was assessed further in all preparations, except those for frozen section, by embedding the muscle in plastic and examining it in whole mount. Due to the thin nature of the cutaneous pectoris muscle it was possible to examine individual axons for disruption of myelin sheaths. Seven muscles were excluded from analysis because a number of axons appeared damaged. The detection of MEPPs in functionally denervated muscles further confirmed that innervation was intact. Also, the absence of a denervated pattern of matrix remodeling in functionally denervated muscles indicated that the innervation to muscles examined by frozen section was intact. Thus in the muscle preparations analyzed, muscle activity was effectively blocked and the innervating nerve was undamaged.
Further evidence for the effectiveness of the activity blockade was the induction of denervation responses in inactive muscle fibers. We demonstrated for the first time a role for muscle activity in inducing denervation responses of frog muscle. Several denervation responses of mammalian mus-cle, including atrophy (20); the appearance of AChRs and neural cell adhesion molecule in extrajunctional regions of the muscle membrane (3,9,12,23,29,41); fibrillations (37); increased expression of Thy-1 (4); and altered expression of the genes coding for voltage-dependent sodium channels (50), the c~, B, tS, and 3' AChR subunits (18), MyoD, and myogenin (14) have been shown to be induced in innervated muscle by activity blockade or reversed in denervated muscle by restoration of activity. We have documented a role for muscle activity in inducing the denervation-like redistribution of two synaptic molecules in frog muscle, AChR and the mAb 3B6 antigen. Extrajunctional sensitivity to ACh was detected in muscle fibers functionally denervated for 3 wk. The detection of MEPPs and/or the presence of intact axons confirmed that the extrajunctional sensitivity to ACh was not a result of axon damage. The extrajunctional ACh sensitivity of denervated frog muscle fibers was similar to previously determined values (13) and comparable sensitivities were obtained from extrajunctional regions of functionally denervated muscle fibers. Loss of muscle activity also resulted in the extrajunctional redistribution of the synaptic antigen mAb 3B6. Its distribution resembles that of AChR in that mAb 3B6 is localized at endplates of innervated muscle fibers but is found extrajunctionally in denervated muscle fibers (7). Thus both AChR and mAb 3B6 appeared in extrajunctional regions of innervated muscle fibers functionally denervated by tetrodotoxin.
One striking feature of this muscle remodeling was that the redistribution of these synaptic molecules occurred more rapidly when induced by denervation than by presynaptic blockade of muscle activity. While extrajunctional ACh potentials were recorded in muscle fibers denervated for 2 wk, 3 wk of functional denervation were required to detect extrajunctional ACh sensitivity. The extrajunctional appearance of the mAb 3B6 antigen was similarly slowed; 3-4 wk of functional inactivity were required to produce the 2 wk denervation response (7). The development of extrajunctional ACh sensitivity in mammalian muscle occurs with a similar time course when induced by either denervation or blockade of synaptic transmission (3,29). Some membrane properties of muscle fibers however are more effectively altered by denervation than by presynaptic blockade of muscle activity including the onset of increased AChR turnover (2). Thus denervation responses of both frog and mammalian muscle fibers are induced by muscle inactivity, however additional signals are likely to contribute as well. Proposed activity-independent signals include loss of spontaneous vesicular release and products of nerve degeneration (2,23,32).
We determined the role of muscle inactivity as a signal in initiating connective tissue remodeling at synaptic sites. The junctional interstitial cell accumulation can be detected one week after nerve damage with a maximal response at 2-3 wk of denervation (6). Given that the onset of this denervation response may be slowed in functionally denervated muscles, we examined interstitial cell density after 2-4 wk of inactivity. Presynaptic blockade of muscle activity in innervated muscles for 2--4 wk had no effect on the distribution of interstitial cells in junctional and extrajunctional regions of muscle. Marked junctional accumulations of interstitial cells were detected in muscles made inactive for similar periods of time by denervation. TTX itself did not prevent the ac-cumulation of interstitial cells since there were junctional accumulations in denervated muscles exposed to TTX. Taken together, these results indicate that the junctional accumulation of interstitial cells in denervated muscle is not initiated by the loss of muscle activity. This is consistent with the observation of Murray and Robbins (34) that blockade of synaptic transmission by tetrodotoxin did not induce a generalized mitotic response in mouse muscle.
An additional consequence of denervation in frog as in mammalian muscle is that tenascin and fibronectin stain become concentrated in the interstitial spaces of junctional regions of muscle. This denervation-like remodeling of the extracellular matrix did not occur in junctional regions of muscles functionally denervated for up to 4 wk. These results indicate that, like the junctional interstitial cell accumulation, molecular remodeling of the muscle matrix is not induced by blockade of muscle activity. Further, these data support the hypothesis that the interstitial cells are responsible for the molecular remodeling of the extracellular matrix in denervated muscle. Interstitial cells isolated from rat muscle produce fibronectin, tenascin, neural cell adhesion molecule, and heparan sulfate proteoglycan in vitro (16). Though a denervation-like accumulation of tenascin was not present in functionally denervated muscles, there was an altered pattern of tenascin immunoreactivity when compared to control innervated muscles. Structures within the connective tissue, presumably nerves, and neuromuscular junctions were more frequently stained by antibodies directed against tenascin in functionally denervated muscles. This result is consistent with the observation that Schwann cells and cells of the perineurium produce tenascin after nerve injury (10), and suggests that this response may be dependent on nerve activity. Thus the production of tenascin in denervated muscle may well arise from different cell sources; Schwann cells, skeletal muscle and fibroblasts produce tenascin (10,16). We propose that the majority of interstitial tenascin in denervated muscles is contributed by the accumulated interstitial cells and is induced only by denervation and not loss of synaptic activity. Some modification of the extracellular matrix in junctional regions of muscle is known to occur with loss of muscle activity. The appearance of neural cell adhesion molecule in the interstitial space, as well as on the muscle fiber surface, is induced by presynaptic blockade of muscle activity with tetrodotoxin (9). Also in rat, the level of tenascin immunoreactivity was increased in functionally denervated muscles, however it was not associated with junctional regions of the muscle (46). Thus denervation may result in the generation of activityindependent signals that induce the remodeling of the junctional connective tissue and act in concert with muscle inactivation to elicit the full denervation response of muscle.
We conclude that the cellular and molecular remodeling of the junctional connective tissue in denervated frog muscle is not mediated by the level of muscle activity. What signal then, other than loss of muscle activity, may be responsible for initiating this connective tissue remodeling after denervation? Previous experiments revealed that the junctional accumulation of interstitial cells involves mitosis (6,16). Thus, damage to motor axons could result in either the production of a positive mitogenic signal that initiates the interstitial cell proliferation and matrix remodeling or the cessation or removal of a negative signal that inhibits interstitial cell proliferation in innervated muscle. Possible cells or structures that may generate the signal include axonal degeneration products, the terminal Schwann cell, the intact nerve terminal, or the muscle fiber. Previously we determined that axonal debris is not the signal; reinnervation of a muscle following nerve crush inhibited the interstitial cell accumulation though axonal debris was present (6). Products of axonal degeneration however have been thought to induce denervation responses on muscle fibers (23), and include molecules such as nerve growth factor, interleukin-1, and apolipoproteins produced following nerve injury (43). The restriction of the connective tissue remodeling to the vicinity of synaptic sites suggests a localized site for signal generation, and thus the terminal Schwann cell, at the neuromuscular junction, is a likely candidate. Though recent evidence indicates that terminal Schwann cells respond to evoked activity of axons (17,22,38), the experiments presented here exclude the possibility of the signal originating in terminal Schwann cells as a consequence of loss of activation. However, phagocytosis of axons by terminal Schwarm cells could induce the production of a signal by these cells and its restriction to the junctional regions of muscle. Terminal Schwann cells at denervated neuromuscular junctions are known to extend elaborate processes (40). In addition, nonglial cells such as macrophages may be involved in the signaling mechanism in response to their phagocytosis of degenerating axons (36). The axon itself remains a candidate given the inhibition of the interstitial cell accumulation by reinnervation of the muscle. Though evoked vesicular release is blocked by TTX, spontaneous release remains intact and the products of release could act on terminal Schwann cells or muscle fibers. Finally, the muscle fiber may be involved in signaling by an activity-independent mechanism. Insulin-like growth factors, one of several factors released by denervated muscle fibers (19), have been shown to induce some degree of connective tissue remodeling (5).
Clearly, further experiments are needed to elucidate the mechanism by which connective tissue remodeling is initiated after denervation. Such information will aid in our determination of the role of the interstitial cells in the regeneration of the neuromuscular junction. The reconfiguration of the junctional matrix after denervation may serve as a favorable substrate for regenerating axons (8,19). We have discovered that interstitial cells are also concentrated in junctional regions of developing muscles during the period of synaptogenesis (Connor, E. A., manuscript in preparation). The similarity in the cellular composition of connective tissue of developing and denervated muscle suggests a role of the interstitial cells in the development of the neuromuscular junction. Further, there is evidence that interstitial ceils near developing neuromuscular junctions are different from those in extrajunctional regions in that they, along with Schwann cells and perineurial cells, express a particular transgene during embryonic and early postnatal life (49). To fully understand the process of synaptogenesis in developing and regenerating systems it is necessary to determine the contribution of the interstitial cells and the matrix molecules that they produce.
We wish to thank R. Murphey for the use of an electrophysiology recording setup, his assistance in those experiments, and comments on the manuscript. We thank V. Budnik, A. Dunaevsky, R. Parsons, and M. Smith for their helpful comments. We also thank D. David for technical assistance.


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Evidence for a functional interaction between the beta chain of major histocompatibility complex class II and the T cell receptor alpha chain during recognition of a bacterial superantigen.

Several studies have suggested that there is a direct interaction between the T cell receptor (TCR) and the major histocompatibility complex (MHC) molecule during T cell recognition of superantigen. To further investigate this possibility, we have analyzed T cell recognition of a bacterial superantigen, Staphylococcal enterotoxin B (SEB), presented by a series of mutant murine I-Ek molecules in which residues of either the alpha or beta chain predicted to interact with the TCR have been substituted. Individual T cell hybridomas gave distinct patterns of responsiveness to SEB presented by the I-E beta k mutants that could not be attributed to differences in the binding of SEB to the mutants. This effect appeared to be dependent on the TCR- alpha chain because some of these hybridomas expressed identical TCR transgenic beta chains. In contrast, none of the hybridomas gave distinct patterns of responsiveness to SEB presented by the I-E alpha k mutants. Taken together, these observations support the idea that there is a functional interaction between the alpha chain of the TCR and the beta chain of the MHC class II molecule. The data also support the idea that this interaction might enhance superantigen recognition in some cases.

Sunlnlary Several studies have suggested that there is a direct interaction between the T cell receptor (TCR) and the major histocompatibility complex (MHC) molecule during T cell recognition of superantigen. To further investigate this possibility, we have analyzed T cell recognition of a bacterial superantigen, Staphylococcal enterotoxin B (SEB), presented by a series of mutant murine I-E k molecules in which residues of either the c~ or ~ chain predicted to interact with the TCR have been substituted. Individual T cell hybridomas gave distinct patterns of responsiveness to SEB presented by the I-EB k mutants that could not be attributed to differences in the binding of SEB to the mutants. This effect appeared to be dependent on the TCR-c~ chain because some of these hybridomas expressed identical TCR transgenic 3 chains. In contrast, none of the hybridomas gave distinct patterns of responsiveness to SEB presented by the I-Eot k mutants. Taken together, these observations support the idea that there is a functional interaction between the ot chain of the TCR and the 3 chain of the MHC class II molecule. The data also support the idea that this interaction might enhance superantigen recognition in some cases. S uperantigens are characterized by their ability to stimulate high frequencies of T cells expressing particular V/~ elements. A current model for superantigen activation of T cells suggests that the superantigen interacts directly with the class II molecule and the TCR V3 element, resulting in cross-linking of the two molecules (1). In addition, there is a growing body of evidence to suggest that there is a direct interaction between the MHC class II molecule and the TCR during superantigen engagement that can influence superantigen-mediated activation of some T ceils (2). First, dements of the TCR-ot chain have been shown to affect the ability of T cells to respond to superantigens (3)(4)(5)(6). Second, it has been shown that MHC polymorphisms can also affect the ability of T cell lines and hybridomas to be stimulated by viral and bacterial superantigens (4,7,8). These effects were not due to the inability of the superantigens to bind to the class II molecules but rather were the result of preferential recognition of superantigen-MHC haplotype complexes by the T cells. Third, mutational and structural analysis of the bacterial superantigen Staphylococcal enterotoxin B (SEB) suggests that the TCR and MHC molecules are brought into dose proximity during superantigen engagement (9)(10)(11).
In the current study, we investigated the hypothesis that TCR-MHC interactions modulate superantigen reactivity by testing the ability of wild-type and mutant I-E k molecules to present the bacterial superantigen, SEB, to T cells.

Materials and Methods
I-E~-expressing Cell Lines. The Chinese hamster ovary (CHO) transfectants expressing I-E ~ wild-type and mutant molecules have been described previously (12). The cells were stained with the anti-I-E k mAb Y17 (13)  Hybridoma Reactivity to SEB, Hybridoma reactivity was determined by a standard Ib2 assay as described previously (7,14). Briefly, 105 hybridomas were cultured with 105 CHO transfectants in 250 gl cultures in complete medium. Supematants were removed after 24 h and assayed for the ability to support the growth of IL-2-dependent HT-2 cells. SEB dose-response curves were performed for each hybridoma and the concentration of SEB required to stimulate IL-2 secretion half-maximally was determined. To normalize the results, the response of the hybridomas to SEB presented by the mutant I-E k molecules was compared with the response with wild-type I-E kpresenting cells, which was given an arbitrary value of 1 (termed relative responses). Each dose-response curve was performed a minimum of three times and the relative responses averaged, and expressed as the mean __ SE. Commercial SEB was purchased from Sigma Chemical Co. (St. Louis, MO) and the recombinant SEB plasmid was a kind gift of Dr. John Kappler (National Jewish Center for Immunology, Denver, CO). Recombinant SEB was prepared as described by Kappler et al. (9).
In some experiments, the CHO transfectants were fixed with 1% paraformaldehyde for 30 min at room temperature. The cells were washed three times with complete medium and used in a standard IL-2 assay as described above.
SEB Biotinylation and Binding Assay. SEB binding to I-E k CHO transfectants was measured using biotinylated SEB. Biotinylation of SEB was carried out using sulfosuccinimidyl-6-(biotinamido) (NHS-LC-biotin) (Pierce, Rockford, IL) and a modification of the manufacturer's protocol. Briefly, SEB was dissolved in 50 mM sodium bicarbonate buffer (pH 8.5). NHS-LC-biotin was dissolved in ddH20 and mixed with SEB at a molar ratio of 160:1 biotin/SEB and incubated at room temperature for 30 min. Free biotin was removed and the buffer changed to lx PBS by repeated centrifugations using Centricon-10 concentrators (Amicon Corp., Beverly, MA). These conditions resulted in the incorporation of four biotin molecules per SEB molecule as determined using avidin (Pierce) and Immunopure 2-(4'-hydroxyazobenzene) benzoic acid (HABA) (Pierce) following the instructions prescribed by the manufacturer.
Binding studies were performed by incubating biotinylated SEB (at concentrations ranging from 0.23 to 15 #M) with CHO transfectants (4 x 105 cells/well) for 2 h on ice. Samples were washed three times with staining buffer (lx PBS, 0.1% sodium azide, 1% FCS), PE coupled to strepavidin (TAGO Inc., Burlingame, CA) was added and samples incubated for 20 min on ice. The cells were washed twice with staining buffer and analyzed on a FACScan | (Becton Dickinson & Co., Mountain View, CA). Fluorescence intensity was calculated by subtracting the fluorescence signal obtained by staining a class II negative CHO transfectant with biotinylated SEB from the fluorescence signal obtained from the I-E k positive cell lines. The background fluorescence signals of the class II negative CHO transfectant ranged between a mean fluorescence intensity of 3.0-4.0.

Results and Discussion
Previous studies have suggested that there is a direct interaction between the TCR and MHC molecules during the recognition of both retroviral and bacterial superantigens (reviewed in 2). One prediction of this model is that mutation of residues on the top face of the class II molecule which are accessible to the TCR would influence superantigen recognition. To test this hypothesis we used 12 I-Ek mutants expressed in CHO cells, previously characterized by Ehrich et al. (12) (Table 1). Each of the mutations introduced a single amino acid substitution in a residue predicted to be involved in TCR-MHC interactions during conventional peptide- " CHO transfectants have been described previously (12). Each cell line expresses I-E k molecules that have single amino acid substitutions in either the oe or the/3 chain. Amino acid changes are shown by the single letter code.
MHC recognition (15). The substituted amino acids were of similar hydrophobicity to the wild-type residue but either had an opposite charge or a bulkier side chain. Therefore, secondary and tertiary structures of the MHC molecule were maintained while promoting maximum disruption of potential TCR-MHC contact points. Consistent with this prediction, the wild-type and mutant I-Ek-expressing CHO cells were shown to be indistinguishable in their ability to bind peptides and anti-I-E antibodies (12). In addition, all the mutants, except 369 and 381, were found to bind the bacterial superantigen SEA normally (12).
To determine the effects of I-E k mutations on bacterial superantigen SEB recognition, we first tested the transfectants for their ability to bind SEB. Direct binding studies revealed that the I-Eot k mutants bound biotinylated SEB comparable with wild-type I-E k molecules (Fig. 1 A). There was a 5-10fold range of SEB binding to the I-E/3 k mutants (Fig. 1 B). Interestingly, the mutation at position 81 in the I-E k/3 chain did not affect SEB binding. This residue has been defined as critical for SEA binding to both the HLA DR1 (16,17) and MHC I-E k molecule (12). Thus, these data are consistent with the idea that SEB and SEA bind to different regions of MHC class II (reviewed in 18).
The I-E k mutants were used to present SEB to a panel of 26 V/38 + T cell hybridomas (4 V/38.1 +, 13/3 chain transgenic [TG] V/38.1 +, and 9 V/38.2 + hybridomas). Doseresponse curves were generated for each hybridoma to SEB presented by each of the I-E k mutants and wild-type I-E k. Figs. 2 and 3 show the relative responses of six representative hybridomas (590-V/38.1 +; 1230, 1345, 1357-TG V/38.1 +; 577, 610-V/38.2+). In each case, the data are presented as the relative shift in the dose response curve of each mutant compared with the wild-type I-E k control.
Five important observations can be drawn from these data. First, all six of the hybridomas shown expressed distinct patterns of reactivity to the I-E k/3 chain mutants. For example, hybridomas 590 and 1230 gave a reciprocal pattern of responsiveness to SEB presented by mutants/369,/377, and/384 (  responded to/377. Although/577 bound SEB more weakly than wild-type I-E k ( Fig. 1 B), /577 can present SEB well to four out of six hybridomas (Fig. 2). Several other distinct patterns of reactivity were observed among the hybridomas suggesting that fine specificity for SEB was a characteristic of the TCR and not SEB binding. These patterns of reactivity were extremely reproducible as shown by minimal variations in these experiments. Moreover, identical patterns were obtained using CHO transfectants that had been fixed in paraformaldehyde and by using either recombinant SEB or commercial preparations of SEB (data not shown). Thus the distinct patterns of SEB recognition by the hybridomas cannot readily be explained by processing of SEB into peptides, contaminants within the commercial SEB preparation or differential binding of SEB to the various I-E~ k mutants (see Fig.  1 B). In addition, the reciprocal patterns of SEB recognition rule out the possibility that the expression of different adhesion molecules on the hybridomas was responsible for their reactivity. Therefore, these data strongly support the hypothesis that there is a direct TCR-MHC interaction during SEB recognition, which can influence T cell activation. Second, distinct patterns of SEB reactivity were observed between hybridomas which expressed identical transgenic TCR-/5 chains (hybridomas 1230, 1345, and 1357). For example, hybridoma 1230 responded well to SEB presented by 369 but weakly to 377, whereas 1357 responded well to SEB presented by/577 but not to/569. Therefore, these data suggest that residues on the a chain of the TCR interact with residues on the/5 chain of the MHC class II molecule. This hypothesis is consistent with the findings of several groups (3-6) that the c~ chain of the TCR can have a strong influence on the recognition of both SEB and the viral superantigen, Mls-1, and is supported by recent crystallographic analysis of SEB bound to the human Dtkl molecule (11). It is interesting to note that the mutation of 369 consistently reduced the response ofaU 17 V/58.1 hybridomas tested irrespective of the TCR-ot chain ( Fig. 2 and data not shown). This observation suggests that residue/569 of the MHC might contact conserved residues of the TCR-~ chain framework region. We are currently analyzing TCR-ot chain usage in these TG hybridomas. Third, some of the hybridomas responded more strongly to SEB presented by mutant I-E ~ molecules than presented by wild-type I-EL For example, 590 gave a 10-fold stronger response to SEB presented on/373 and 1377 compared with presentation by wild-type I-E k. Interestingly, these mutations at positions 1373 (A-~V) and/377 (T--~Q) increase the size of the side chain groups. In mutagenesis studies of protein-protein interactions it is not uncommon to find that an increase in side chain size (or a charge reversal) can enhance interactions. This observation is consistent with the findings that bulky side chains can enhance TCR-MHC-antigen recognition (12). The increased responsiveness seen with these mutations was not due to increased SEB binding (Fig. 1 B) and was not a characteristic of all hybridomas (for example, 1345 responses were 10-fold weaker to SEB presented by these mutants compared with wild-type).
Fourth, most of the V/38.2 + hybridomas responded similarly to all I-E k mutants (610, Fig. 2 F, and data not shown). It is interesting to note that V/38.2 + T cells are thought to have a higher affinity for SEB than V/38.1 + T cells (7), consistent with the hypothesis that TCRs with a high affinity for superantigen are less influenced by TCR-MHC interactions (2). In support of this hypothesis, the VB8.2 + hybridomas studied here generally responded to nanomolar amounts of SEB, whereas the V/38.1 + hybridomas required micromolar amounts of SEB (data not shown). However, we did identify one V/38.2 + hybridoma, 577 (Fig. 2 E), with a distinct pattern of SEB recognition. This observation demon-strates that TCR-MHC interactions can also influence strongly SEB-reactive hybridomas.
Fifth, in contrast to the I-E k/3 chain mutants, recognition of the I-E k c~ chain mutants did not reveal distinct patterns of fine specificity among the hybridomas (Fig. 3). Although individual mutants appeared to be stronger (for example, ot61) or weaker (for example, ot57, 65, 68, 72, 79) presenters of SEB, all hybridomas gave essentially the same pattern of SEB recognition. These differences did not correlate with binding of SEB. For example, ot61 did not bind SEB better than the other mutants ( Fig. 1 A). Therefore, the data obtained from analysis of these I-E k ot chain mutations provides no evidence for interaction between the TCR and the MHC ot chain. This is consistent with the crystallographic analysis of the HLA DR1/SEB complex which indicates that much of the MHC c~ chain is buried under the SEB molecule and is presumably not able to contact TCR (11). The observation that mutation of the I-E k a chain at position 61 considerably enhanced SEB reactivity is interesting in this regard. Labrecque et al. (8) have proposed that class II molecules may induce a conformational change on bound bacterial superantigens which is necessary for TCR recognition (based upon the study by Gascoigne and Ames [19]). The generic effect of I-E k a chain mutations, specifically a61, is consistent with an effect on the structural conformation of bound SEB.
Taken together, the data presented show that mutations within the I-E k 13 chain but not the I-E k ot chain, resulted in unique patterns of SEB recognition by V/38 + T cells. These data are fully consistent with the hypothesis that residues within the MHC class II 13 chain contact the TCR and can influence superantig~n-mediated T cell activation. Furthermore, a comparison of T cell responses between hybridomas expressing transgenic fl chain TCRs suggests that the TCR-cx chain is involved in contacting the fl chain of the MHC. This hypothesis is consistent with the TCR-MHC orientation predicted by the recent crystallographic studies of DR1-SEB complexes (11).