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Polymorphonuclear neutrophils express the common acute lymphoblastic leukemia antigen.

Monoclonal antibodies J5, VIL-A1, and BA-3, known to react with the common acute lymphoblastic leukemia antigen (CALLA) were found to specifically stain normal human polymorphonuclear neutrophils (PMN). The antigen detected on PMN had a molecular weight (95,000-110,000 mol wt) close to that of CALLA (95,000-100,000 mol wt) and thus these surface membrane antigens are likely related, if not identical. The fluorescent staining intensity of PMN is comparable to that of CALLA- positive leukemic cells and the presence of PMN in patient samples could potentially produce false-positive results in diagnosis.


Materials and Methods
Cells. PMN were isolated from the peripheral blood of 13 healthy normal adult volunteers. The granulocyte-and erythrocyte-rich fraction was separated by gradient centrifugation with Ficoll-Hypaque (Accurate Chemical & Scientific Corp., Westbury, NY) (600 g, 30 min) then mixed 1:1 with Plasma-Gel (HTI Corporation, Buffalo, NY) for 30 min at 37°C to allow the erythrocytes to settle out. The granulocyte-rich supernatant was washed three times in medium RPMI 1640 (Gibco Laboratories, Grand Island Biological Co., Grand Island, NY) and resuspended at 3 × 10*/ml. Purity of the preparations was documented morphologically on cytocentrifuge preparations. Cell lines studied included Raji (Burkitt's lymphoma), NALM-1 (lymphoblast crisis of chronic myelogenous leukemia), MOLT-4 (T ALL), and HL60 (promyelocytic leukemia). The induction of HL60 cells to more mature myeloid cells was performed with dimethyl sulfoxide (DMSO) (1.25%; Microbiological Associates, Walkersville, MD) and 12-tetradencanoyl-phorbol-13-acetate (TPA) (10 -s M; Sigma Chemical Co., St. Louis, MO) as previously described (9,10). Cell suspensions were prepared from lymph nodes involved by follicular lymphoma (12 cases) by pressing teased-out cells through a stainless steel mesh and washing in RPMI 1640.

Results
The granulocyte fraction from all normal individuals tested stained with J5, VIL-A1, and BA-3 (mean 89%, range 55-99%). All three preparations of J5, namely, ascites, intact IgG, and F(ab')2, reacted with granulocytes ( Fig. 1 A, B). Greater than 90% of cells stained with OKM1, however, no fluorescence was observed with the nonreactive mouse ascites, purified murine IgG1, IgG2a, IgG2b, IgG3, antibodies OKT6 and Leu-1, or after omission of primary antibody. Further, preincubation of cells with normal human serum or human IgG1 did not affect either the percentage of cells stained by J5 or the intensity of fluorescence (Fig. 1 C). To determine whether anti-CALLA antibody could distinguish between neutrophils and eosinophils, the leukocytes from normal individuals and from a patient with hypereosinophilia were stained with the F(ab')2 fraction of J5 and sorted into fluorescent-positive and -negative fractions on the FACS. The cells were collected, cytocentrifuged, stained with Wright's stain, and differential counts were performed. The positive fractions were greatly enriched for PMN (95 and 100% for normals and 86% for the hypereosinophilia patient), whereas the negative fractions were predominantly eosinophils, lymphocytes, and monocytes. The fluorescence intensity of neutrophils stained with j5 was comparable to that observed with follicular lymphoma cells but less than that found with Raji cells (mean channel 160) and NALM-1 cells (mean channel 180) ( Fig. 1 D). The average mean fluorescence channel was 83 for PMN (10 samples) and 102 for follicular lymphomas (12 samples). IgG-J5 also reacted with HL60; however, this interaction appeared to be mediated through Fc receptors since (a) staining was inhibited by preincubation with human serum or human IgG1 myeloma protein, and (b) F(ab')2 J5 did not react with HL60. Addition of either TPA or DMSO to HL60 cells did not result in induction of specific J5 reactivity during a 5-d course.
The membrane determinant detected on PMN by anti-CALLA antibodies was biochemically characterized by cell surface labeling, immunoprecipitation, and SDS-PAGE gel electrophoresis. Autoradiographs of these gels demonstrated that J5 antibody identified a protein on the cell surface of PMN of ~95,000-110,000 mol wt, and was similar in size to that found on Raji cells (95,000-100,000 mol wt) (Fig. 2). These proteins recognized by J5 were not present in immunoprecipitates performed with control UPC10 IgG2a monoclonal antibodies or on the T cell leukemia cell line, MOLT-4.

Discussion
These studies demonstrate that the anti-CALLA monoclonal antibodies, J5, VIL-A1, and BA-3 consistently react with human polymorphonuclear neutrophils but not  PMN (a, b), Raji (c, d), and Molt-4 (e, f) were immunoprecipltated with a nonreactive murine IgG2a 0JPC10) (a, c, e) or j5 (b, d, f). From PMN, J5 immunopreeipitated a single band that has a molecular weight similar to that of the band obtained with Raji cells. These proteins were not immunoprecipitated with Molt-4 cells or with UPC 10 monoclonal antibodies.
with other leukocytes. This reactivity does not appear to be mediated by cell surface Fc receptors since staining persisted after saturation of these receptors with either h u m a n IgG1 myeloma protein or normal h u m a n serum. Furthermore, the F(ab')2 fraction and the intact IgG form of J5 showed equivalent reactivity. Other routine IgG2a antibodies, including Leu-1, did not react with granulocytes. Preparative cell sorting of J5-positive and -negative fractions indicated that the J5-positive cells observed a m o n g granulocytes and unseparated peripheral blood leukocytes were, indeed, PMN.
T h e antigen detected on the cell membrane of P M N was similar in size to C A L L A present on Raji cells (95,000-110,000 tool wt for P M N and 95,000-100,000 mol wt for Raji) and was immunostained by three different anti-CALLA antibodies. Thus, the antigen detected on P M N not only shares an epitope with C A L L A , but these antigens m a y be identical. Although blasts from the lymphoblast crisis of chronic myelogenous leukemia (CML) are frequently CALLA-positive (2,4,7), C A L L A may not be expressed on myeloid precursor cells since; (a) the cells of the stable phase of C M L are reported to lack C A L L A (7); (b) anti-CALLA (J5) treatment does not inhibit proliferation in myeloid clonal assays (12); and (c) as observed in the present study, the promyelocytic leukemia cell line, HL60, did not specifically stain with J5. Thus, C A L L A appears to be acquired at a late stage of P M N differentiation. Although a n t i -C contained 6% J5-stained cells (Fig. 3). This result was initially interpreted as indicating a low level of involvement by the neoplastic process. However, when these stained cells were sorted on the FA(3S, they proved to be entirely composed of PMN. Therefore, caution should be exercised when studying effusions, bone marrow aspirates, and especially unseparated leukocyte buffy coats as has been suggested for immunofluorescence analysis by flow cytometry (13).

Summary
Monoclonal antibodies J5, VIL-A1, and BA-3, known to react with the common acute lymphoblastic leukemia antigen (CALLA) were found to specifically stain normal human polymorphonuclear neutrophils (PMN). The antigen detected on PMN had a molecular weight (95,000-110,000 mol wt) close to that of CALLA (95,000-100,000 mol wt) and thus these surface membrane antigens are likely related, if not identical. The fluorescent staining intensity of PMN is comparable to that of CALLA-positive leukemic cells and the presence of PMN in patient samples could potentially produce false-positive results in diagnosis.
Received for publication 13 December 1983.