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Contrasting in vitro vs. in vivo effects of a cell membrane-specific CC-chemokine binding protein on macrophage chemotaxis

Abstract Chemokines (CK) provide directional cues that mediate the recruitment of leukocytes to sites of inflammation. Broad-spectrum blockade of the CC-CK family, using the vaccinia virus 35K protein, has been shown to cause a potent reduction of systemic inflammation in models of atherosclerosis, vein graft disease and arthritis. We have used a cell membrane-targeted form of 35K, Mem35K, to probe whether cell-associated blockade of chemokine response is sufficient to reduce cell recruitment in inflammation. In Tie2cre mice, activation of a flox-stop Mem35K transgene directed conditional expression of Mem35K in leukocytes and endothelial cells, confirmed by Western blotting, flow cytometry and immunofluorescence microscopy. This conditional Mem35K expression was sufficient to increase cell surface CCL5 binding and reduce chemotaxis in vitro to CCL5, CCL2 and CCL3 but not to non-CC-CK chemoattractants, LTB4, C5a or chemerin. However, in vivo monocyte recruitment into the peritoneum driven by zymosan or CC-chemokine injection, which was demonstrated to be CC-CK dependent using CCR2−/− mice, was not reduced by Mem35K expression, despite the expression of functional Mem35K protein. These findings highlight differing requirements for cell-associated anti-inflammatory activity in in vitro and in vivo models. Key message Mem35K is a cell-associated CC-chemokine binding protein. Conditional Mem35K transgenic mice show expression Mem35K in leukocytes. Mem35K blocks in vitro primary macrophage chemotaxis specifically towards CC-chemokines. Mem35K expression is not sufficient to reduce inflammation in vivo. The requirements for anti-inflammatory activity in vitro and in vivo are different. Electronic supplementary material The online version of this article (doi:10.1007/s00109-014-1194-6) contains supplementary material, which is available to authorized users.


Breeding Mem35K transgenic and Tie2cre transgenic mice
The Mem35K transgene is inserted into the X chromosome, therefore female heterozygous mice manifest mosaic expression, due to random X inactivation. Due to this phenomenon only hemizygous male and homozygous female Mem35K mice are used for experiments. No gene dosage effect is present as both male and female mice will only express Mem35K from one allele. Initial characterization was carried out on C57Bl/6J backcross generation 3 animals, with key findings such as peritonitis and chemotaxis data reproduced on backcross 6 animals. The Tie2cre transgene is active in the female germline, as such only male animals are used to establish breeding pairs to maintain conditional expression. Mice were genotyped using primers targeted against the wildtype HPRT allele, floxed sequence and in a separate reaction for the presence of the cre sequence.

Genomic DNA production and excision PCR
Genomic DNA for detection of the excised allele was produced using the Qiamp kit (Qiagen).The floxed and excised allele were detected using the following primers: The floxed allele yields a 536bp product and the excised allele a product of 876bp.

Primary cell preparations:
Bio-Gel Elicitation of Primary Mouse Macrophages.

Blood, Bone Marrow and Spleen Leukocytes
Single cell suspensions of splenocytes and bone marrow cells were obtained using standard protocols (1). Blood samples were taken directly into an EDTA coated tube (Teklab). All cell populations were stained with monoclonal antibodies directed against CD3, B220, Ly--6G (all BD Biosciences) and the 7/4 antigen (AbD Serotech). The total cell population was gated by forward scatter and side scatter then interrogated for expression of the relevant cells markers compared to isotype controls (T--cells -CD3 + , B--Cells -B220 + , Neutrophils -7/4 HI , Ly--6G + or Monocytes -7/4 HI , Ly--6G --). Cells were enumerated by an absolute count as described below.

Flow cytometry
All flow cytometry was performed using a DAKO CyAn cytometer and Summit software (both Beckton Coulter). Data was analysed using Flow Jo software (TreeStar Inc).
Where cells were enumerated an absolute count protocol was used where cells were quantifed by ratio to a known number of fluorescent beads spiked into the sample prior to analysis ( (2)). For quantification of fluorescence the geometric mean fluorescence of the histogram population was used.
All confocal microscopy was performed using a Zeiss confocal with a 63x oil immersion objective and LSM software with multi--track acquisition setup.

cell CCR5-mediated chemotaxis
CCR5 mediated chemotaxis was assessed using 293 cells transfected with CCR5, EGFP and mem35K (or empty plasmid control) in Chemotx plates (Neuroprobe) as described previously (3). Each experimental sample was analyzed in triplicate, and three separate images were quantified for each membrane.

CCL5 binding assay and CCR5 expression.
Binding of human CCL5 to Biogel--elicited macrophages was assessed using a Fluorokine CCL5 assay, according to the manufacturer's instructions (R&D Systems). Macrophages were identified as FSC HI /SSC HI cells. Parallel cell isolations were used to assess cell surface CCR5 expression by flow cytometry using an anti--CCR5 antibody (BD Biosciences). CCL5 binding, CCR5 expression and endogenous GFP fluorescence was assessed in n=3--6 animals per genotype and significant alterations in any parameter were assessed by T--test, with p<0.05 being judged statistically significant.

xCELLigence real-time Chemotaxis assay
Biogel elicited cells were subjected to chemotaxis assays in CIM--16 well plates using an xCELLigence RTCA--DP instrument as described previously ((4)). Briefly, agonists were loaded into the lower chamber of the CIM--16 plate and the upper chamber attached.
The electrode embedded membrane was equilibrated with prewarmed buffer for 30 mins prior to addition of 8x10 6 Biogel--elicited cells. Migration was assessed every 5s for 4 hours in total. Data were normalized to wells that received media alone and analysis of area under the curve was carried out with RCTA Software version 1.2.1.

Peritonitis models
Zymosan--induced peritonitis was performed as described previously ((5)). In brief, mice were injected intraperitoneally with 10 μg or 100 μg of zymosan A (Sigma--Aldrich) or 2ug JE (Peprotech EC) diluted in 0.5 ml of PBS or PBS alone. Four hours (JE and 10μg zymosan) or 16 hours (100μg zymosan) later mice were sacrificed and the peritoneal cavity was lavaged with 5 ml of PBS/2 mM EDTA. Peritoneal exudate cells were stained with antibodies against Ly6G (BD Biosciences,) and 7/4 (AbD Serotec) and analyzed by flow cytometry to assess neutrophil and monocyte recruitment.

Statistics
All statistical analyses were performed using Prism software version 5 (Graph Pad). All values are expressed as the mean ± SEM. Data were analyzed using a two--way analysis of variance and the Bonferroni post hoc test of significance was used to test between treatment groups at individual dose or time points if appropriate. Where a single dose or timepoint was analysed a one--way ANOVA was used. A value of P<0.05 was the criterion of significance. (C) To more directly assess CC chemokine mediated cell recruitment mice were injected ip with 2µg CCL2 and peritoneal lavage was performed after 4 hours and neutrophils counted. (n=3-6 per group, * p<0.05 by T-test). n=1-2 saline injected animals included to confirm an absence of pre-existing inflammation. Lack of CCR2 caused a small but significant decrease in neutrophil recruitment to 100ug at 16 hours after injection, but not to any other inflammatory stimulus injected. The neutrophil response to CCL2 injection was not ablated in the absence of CCR2 indicating neutrophil recruitment is likely a result of contaminants in the CCL2 preparation.