Binding and Immunoprecipitation (Phytohemagglutinin/HLA-DR Antigens/HLA-A, -B Antigens) JORDAN S
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Proc. NatL Acad. Sci. USA Vol. 79, pp. 6641-6645, November 1982 Immunology Expression of Ia-like antigens by human vascular endothelial cells is inducible in vitro: Demonstration by monoclonal antibody binding and immunoprecipitation (phytohemagglutinin/HLA-DR antigens/HLA-A, -B antigens) JORDAN S. POBER AND MICHAEL A. GIMBRONE, JR. Laboratory ofVascular Pathophysiology, Departments of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 Communicated by Baruj Benacerraf, July 6, 1982 ABSTRACT The expression of Ia-like antigens by cultured clonal antibodies has suggested that endothelial cells may lack human endothelial cells has been investigated by means ofmono- Ia-like antigens (16). clonal antibody binding to intact cells and by immunoprecipitation The expression of HLA-DR or other Ia-like antigens by vas- ofradioiodinated membrane proteins. Primary growing and con- cular endothelium would have at least two important biological fluent cultures of human umbilical vein endothelium express lit- implications. First, the presence of Ia-like antigen-bearing en- tle, ifany, detectable Ia-like antigens under standard culture con- dothelial cells within an organ graft could significantly enhance ditions. However, treatment of primary cultures with the lectin its immunogenicity and hence its likelihood of transplant re- phytohemagglutinin induces the expression of Ia-like antigens. jection (17). In the mixed reaction, a model oftrans- This action of the lectin uniformly affects all the endothelial cells lymphocyte in a culture, does notdepend on cell division, and is associated with plant rejection, Ia-like antigens on the stimulator lymphocyte a cell shape change. The data presented in this report provide population serve as the major provokers ofallogeneic responder unequivocal serological and biochemical demonstration of Ia-like lymphocyte proliferation (18). Interestingly, cultures ofHUVE antigens on human vascular endothelial cells. The fact that the cells also induce allogeneic lymphocytes to proliferate (19) and expression ofIa-like antigens by endothelium can be induced may a role for endothelial Ia-like molecules has been suggested in have important implications for organ transplantation and for reg- this phenomenon (20). Second, Ia-like antigen-bearing endo- ulation of the immunological response. thelial cells might function as immune accessory cells. Indeed, it has been shown that endothelial cell cultures may replace cells Human class II histocompatibility antigens or Ia-like molecules of the monocyte/macrophage line in supporting both phyto- (e.g. HLA-DR, DC/MB/MT/LB, or SB antigens) are essential hemagglutinin (PHA)-induced proliferation of T lymphocytes for effective communication among cells participating in the (21) and antigen-induced proliferation oflymphocytes in a sec- immunological response (1). In humans and rodents, class II ondary in vitro immunological response (22-24), and evidence antigens are expressed most abundantly by B lymphocytes, ac- has been obtained to support a functional role for endothelial tivated T lymphocytes and antigen-presenting cells, such as Ia-like molecules in antigen presentation. In most antigen-pre- cells of the monocyte/macrophage line, Langerhans cells ofthe sentation experiments, however, minor contamination by skin, and dendritic cells of lymphoid organs (2, 3). Thus, Ia-like known Ia-like antigen-positive immune accessory cells (e.g., antigens have been traditionally associated with immunologi- monocytes or dendritic cells) was not ruled out. Still, the sug- cally active cells. Recent morphological studies, using immu- gestion that endothelium can act as an immune accessory tissue nofluorescence microscopy in frozen tissue sections (4-7) or has raised new possibilities for regulation ofthe immunological antibody binding assays ofdispersed tissue cells (8), suggest that response. human endothelium in some vascular beds also appears to ex- For these reasons, we felt it was important to establish un- press Ia-like antigens. In rat tissues, some workers report the ambiguously whether vascular endothelium expresses Ia-like Ia a recent molecules. We have used binding assays with several recently absence of antigens on endothelium (9, 10), although available mouse monoclonal antibodies reactive with human Ia report indicates that certain subclasses of Ia antigens may be antigens and immunoprecipitation of radioiodine-labeled pro- expressed (11). With such morphological methods, however, teins with a strong xenoserum. We report here that primary Ia-like antigenic positivity could be caused by passive adsorp- cultures of HUVE cells express few, if any, Ia-like molecules tion of antigen from the blood either onto the endothelial sur- under standard culture conditions. However, exposure ofthese face or into the underlying basement membrane. cultures to PHA induces a uniform expression of Ia-like mole- Serological and biochemical studies of isolated human vas- cules by endothelial cells. This inducible expression of Ia-like cular endothelial cells have the potential to clarify whether this molecules by vascular endothelium has important implications tissue does express Ia-like antigens. Cultured human umbilical for organ transplantation and for regulation of the immune vein endothelial (HUVE) cells (12) show some susceptibility to response. cytoxicity by anti-HLA-DR xenosera or allosera, suggesting that they express HLA-DR antigens or other Ia-like molecules MATERIALS AND METHODS (13-15). However, in these studies, the level ofcytotoxicity was HUVE cells were harvested by collagenase treatment of two limited and the anti-human serological reagents available may to six normal-term umbilical cord segments and pooled in me- have contained reactivities toward antigens other than Ia-like dium 199/20% (vol/vol) heat-inactivated fetal bovine serum molecules. Furthermore, a recent binding study using mono- supplemented with penicillin at 125 units/ml, streptomycin at 125 ,ug/ml, and 2 mM L-glutamine (medium A; all components The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: HUVE cells, human umbilical vein endothelial cells; ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. PHA, phvtohemagglutinin; Pi/NaCl, phosphate-buffered saline. 6641 Downloaded by guest on September 27, 2021 6642 Immunology: Pober and Gimbrone Proc. Nad Acad. Sci. USA 79 (1982) from M. A. Bioproducts, Walkersville, MD) as described (11). as assessed with "2I-labeled sheep anti-mouse immunoglobulin Replicate primary cultures were plated (day 0) in either Costar F(ab')2 fragment. Biotin-conjugated horse anti-mouse immu- 96-well microtiter plates or Costar 24-well plates (M. A. Bio- noglobulin and succinylated avidin-biotin-conjugated horse- products). The cultures were washed (day 1) to remove unat- radish peroxidase complex (Vector, Burlingame, CA) were tached blood and endothelial cells and refed with medium A bound in second and third steps, respectively. Horseradish per- containing PHA (from Phaseolus vulgaris, type IV; leucoagglu- oxidase binding was localized by the diaminobenzidine (Sigma) tinin from Sigma) at 0-8 pug/ml. Cultures normally reached reaction followed by light microscopy. confluence (ca. 1 x 105cells per cm2)by day 3 and were assayed For immune precipitation, 1 X 106 lymphoblastoid or non- in different experiments on days 1-5. In some experiments, enzymatically suspended HUVE cells (by 5 mM EDTA in Pi/ PHAwas added after confluence was reached (day 3) rather than NaCValbumin) were washed three times with Dulbecco's Pi/ on day 1. NaCl and radioiodinated by using Iodo-bead catalyst (Pierce) Antibody-binding experiments (25) were carried out at ice in 500 ul of the Pi/NaCl containing 250 ,uCi of Na'25I (1 Ci temperature using ca. 1.5 x 104 endothelial cells in confluent = 3.7 X 1010 becquerels; New England Nuclear) for 15 min at monolayers in Costar 96-well microtiter plates. Each well was room temperature. The reaction was stopped by transferring washed twice with Dulbecco's phosphate-buffered saline (Pi/ the cell suspension to a clean tube without the Iodo-bead. The NaCl) containing 1% bovine serum albumin (fraction V; Sigma). cells were immediately washed three times with Dulbecco's Pi/ Substitution ofheat-inactivated 20% (vol/vol) human serum in NaCl/albumin/10 mM KI. The final cell pellet was extracted Dulbecco's Pi/NaCl instead of1% albumin to block nonspecific with 0.5 ml of 2% Nonidet P-40 (Sigma) in 25 mM NaCI/25 mM binding of mouse immunoglobulin to cellular Fr receptors had TrisHCI, pH 7.4/0.1 mM phenylmethylsulfonyl fluoride no effect on antibody binding to HUVE cells. Each mouse (Sigma) for 30 min at ice temperature and the detergent extract monoclonal antibody (diluted 1:100 from an ascitic fluid) was was clarified by centrifugation. Nonspecifically precipitating added in 100 A1 of Dulbecco's Pi/NaCl/albumin and incubated material was removed by sequential incubation with normal for 1 hr; unbound specific antibody was removed by washing rabbit serum and heat-killed formalin-fixed Staphylococcal A three times with the same saline solution. "2I-Labeled sheep bacteria and centrifugation. Specific precipitations were carried anti-mouse immunoglobulin F(ab')2 fragment (New England out by a modification (31) ofthe method ofKessler (32) from the Nuclear, diluted 1:20 or 1:10) or affinity-purified "2I-labeled residual supernatant