IN VITRO STUDIES OF INFLAMMATORY BOWEL DISEASE

Transcription

1 GASTROENTEROLOGY 70: , 1976 Copyright 1976 b y The Willia ms & Wilkins Co. Vol. 70, No.2 Printed in U.S.A. IN VITRO STUDIES OF INFLAMMATORY BOWEL DISEASE Surface receptors of the mononuclear cell required to lyse allogeneic colonic epithelial cells JOHN D. STOBO, M.D., T. B. TOMASI, M.D., PH.D., K. A. HUIZENGA, M.D., R. J. SPENCER, M.D., AND R. G. SHORTER, M.D. Departments of Immunology, Surgery and Medicine, Mf!Yo Medical School and Foundation, Rochester, Minnesota Peripheral blood mononuclear cells from patients with either Crohn's disease or ulcerative colitis (collectively referred to as inflammatory bowel disease) are cytotoxic in vitro for isologous or allogeneic colonic epithelial cells. Utilizing the ability of thymusderived (T) lymphocytes to bind sheep red blood cells to their surface and the property of bone marrow-derived (B) lymphocytes to display easily detectable surface immunoglobulin determinants, the cytoxicity of these lymphocyte subpopulations was tested. The results indicate that the mononuclear cell required for the lysis of colonic epithelium was not included within the bulk of Tor B lymphocytes. Indeed, enrichment for cytotoxicity correlated best with enrichment for a mononuclear cell population lacking classical T or B markers. Additionally, mononuclear cells specifically adhering to plastic petri dishes coated with heat-aggregated immunoglobulin, and thus presumably bearing a surface Fc receptor, were enriched in their cytotoxicity. Alternatively, cells not adhering to aggregated Ig-coated petri dishes were relatively depleted of cytotoxicity. The implications of these findings as they relate to possible interactions between cellular and humoral immune mechanisms as a pathogenic mechanism for the colonic inflammatory process noted in patients with inflammatory bowel disease are discussed. lates with the clinical severity of the disease and that it diminished after colectomy would support this.6 Although several investigators have described abnormalities of cellular and humoral immunity in patients with Crohn's disease and ulcerative colitis, it has been difficult to implicate these in the pathogenesis of the colonic inflammatory process (for review see Reference 1). For example, while circulating antibodies with specificities for antigens localized to colonic mucosa can be detected in these patients, it has been shown that these antibodies do not lyse, in vitro, colonic epithelial cells and that their circulating titers do not correlate with disease activity.2 However, the demonstration that peripheral blood mononuclear cells from patients with either Crohn's disease or ulcerative colitis (collectively termed inflammatory bowel disease or IBD) can specifically lyse, in vitro, isologous or allogeneic colonic epithelial cells suggests that this in vitro phenomena may have relevance to the in vivo colonic inflammatory process. 3-5 Indeed, the finding that the in vitro cytotoxicity corre- Received June 2, Accepted September 10, This work was supporterl by Public Health Service Grant AI Dr. Stobo is a recipient of a senior investigator award from the American Arthritis Foundation. The authors would like to thank Dr. ~ i c k J. o lcalvanico a s for his generous gift of the Fab fragments of human IgG. They also thank Mr. Terry Murray and Mrs. Sharon Collyer for their superb technical assistance. 171 In an attempt to elucidate the immunological mechanisms involved we have investigated the nature of the mononuclear cells required for this in vitro cytotoxicity. Thus, utilizing the ability of thymus-derived (T) lymphocytes to bind sheep red blood cells (S-RBC) to their surface 7 and the property of bone marrow-derived (B) lymphocytes to display surface immunoglobulin determinants,8 it appears that the cytotoxic cell is not included within the majority of either the T or B cell populations of lymphocytes and may thus belong to the "null" or " K" population of cells. 9 This norfphagocytic cell does, however, bear a surface receptor which is capable of combining to the Fc portion of immunoglobulin. These findings allow certain speculations as to how the cytotoxicity may be generated and may have pertinence to our understanding of the role of cellular and humoral immune mechanisms in mediating the in vivo colonic inflammatory process. Materials and Methods Patients. Twelve patients, 6 with ulcerative colitis and 6 with Crohn's disease as determined by radiological and pathological criteria, were utilized as the study group. All 12 patients manifested active disease at the time of study and one-half of the patients were receiving systemic corticosteroid therapy. In addition, 2 patients with clinically quiescent Crohn's disease and 4 normal laboratory volunteers were used as a control group. Mononuclear cell subpopulations. Peripheral blood lympho-

2 172 STOBO ET AL. Vol. 70, No.2 cytes, containing less than 2% granulocytes, were isolated from patients with IBC by sedimentation of the buffy coat obtained from heparinized blood, over ficoll-hypaque. 1o The frequency of lymphocytes capable of binding S-RBC to their surface was determined by mixing 0.1 ml of lymphocytes suspended at a concentration of 10 to 15 X 10 6 per ml in RPM I 1640 medium (Grand Island Biologic Co., Grand Island, New York) with 0.1 ml of washed S-RBC (5 x 10 8 per mi). Fifty microliters of human AB serum which had been heated at 56 C for 112 hr and absorbed with S-RBC was added, and the mixture incubated at 37 C for 15 min. The cells were then centrifuged at 50 g for 5 min followed by a 2-hr incubation at 4 C. At the end of this time, the cell pellet was gently resuspended by hand and the frequency of small round mononuclear cells binding three or more S-RBC to their surface (rosettes) enumerated by light microscopy. To separate lymphocytes forming rosettes with S-RBC from lymphocytes not binding S-RBC, rosettes were prepared as described above. The cell suspension was then layered over ficoll-hypaque and centrifuged at 250 g for 20 min at 23 C. Cells remaining at the top of ficoll-hypaque as well as cells sedimenting to the bottom of the tube were removed separately and the S-RBC remaining in each layer lysed with Tris-buffered ammonium chloride. 11 Antiserum used to determine the relative frequency of lymphocytes bearing surface immunoglobulin (Ig) determinants, or B cells, was prepared by the repeated immunization of a sheep with the Fab fragments of human IgG obtained by papain digestion of Cohn Fraction II (a generous gift of Dr. N. Calvanico). Antibody containing specificity against both K and A chains was purified from the antiserum by adsorption to and elution from an agarose affinity column to which human IgG had been linked. The antibody was labeled with fluorescein by the dialysis technique of Clark and Sheoard 12 and the frequency of lymphocytes bearing easily detectable Ig determinants enumerated as described previously. IS Populations of mononuclear cells enriched for or depleted of lymphocytes bearing either surface Ig receptors or receptors capable of combining to the Fc portion of Ig were obtained utilizing a modification of the technique described by Choi et al. H Two milligrams of purified anti-ig antibody or 10 mg of aggregated IgG obtained by heating Cohn Fraction II (50 mg per ml in phosphate-buffered saline, ph 7.4) at 63 C for 1/2 hr was added to polystyrene petri dishes for 20 hr at 23 C. During this time, the protein bound noncovalently to the polystyrene. The unbound protein was poured off, the dishes rinsed several times with sterile RPMI 1640 medium, and 5 ml of 1 % bovine serum albumin (BSA) placed in each dish for 2 hr at 23 C. At the end of this time, the excess BSA was removed and the dishes were washed several times with RPMI 1640 medium. Six to eight million lymphocytes, suspended in 3 ml of RPM I 1640 medium (0.01 % sodium azide) were then added to the petri dishes for 40 min at 37 C. Cells not adhering to the dishes were gently pipetted off while adherent cells were scraped from the dish with a rubber policeman. In vitro cytotoxicity assay. The ability of peripheral blood mononuclear cell populations (PBM) to lyse, in vitro, allogenic colonic epithelial cells was tested as previously described. 15 Briefly, 50,000 mononuclear cells suspended in 0.5 ml of serum-free tissue culture medium 199 (T.C. 199, Grand Island Biologic Co., Grand Island, N. Y.) were mixed with 50,000 fresh, trypsinized (to achieve a single cell suspension) colonic epithelial cells. The mixture was incubated at 37 C for 4 hr with continuous rocking. At the end of this time the number of viable colonic epithelial cells, as determined by trypan blue, was calculated and compared to that noted for colonic epithelial cells incubated without peripheral blood mononuclear cells. The percentage of cytotoxicity was then calculated as previously described. 15 Two types of controls were performed. Firstly, PBM enriched for T cells, B cells, or Fc receptor bearing cells were obtained from 4 normal individuals and 2 patients with a clinical remission of their IBC. The ability of these cells to lyse colonic epithelium was tested. In none of the experiments was more than 3% cytotoxicity detected when compared to that noted for epithelial cells cultured alone. Similarly, it has previously been demonstrated that PBM from patients with diverticulitis did not lyse allogeneic colonic epithelial cells. 15 Moreover, the degree of cytotoxicity correlates with the activity of the colonic inflammation and concomitant corticosteroid therapy in the presence of active disease did not significantly alter the in vitro cytotoxicity.15 Secondly, T and B enriched populations from patients with IBD obtained by the differential sedimentation of rosette-forming cells through ficollhypaque, were tested for their ability to lyse gastric and duodenal epithelial cells. The T-enriched populations demonstrated 1 and 3% cytotoxicity for duodenal and gastric epithelial cells respectively, whereas the B enriched populations lysed 3 and 4% of these same target cells. These same T and B populations demonstrated 14 and 21 % cytotoxicity respectively for colonic epithelial cells. Results The ability of human T lymphocytes to bind S-RBC to the surface, thus forming a rosette, not only is useful for quantitating T cells, but also provides a convenient method for their isolation. 7 Peripheral blood mononuclear cells from patients with IBD were mixed with S-RBC so that the T cells could form rosettes. Cells binding S-RBC to their surface were then separated from cells not forming rosettes by sedimentation over ficollhypaque (table 1). S-RBC in the recovered fractions were removed by hypotonic lysis, and each fraction was then assayed for the relative frequency of B cells utilizing a fluoresceinated anti-ig antibody as well as their ability to lyse colonic epithelium. Control populations consisted of cells which were separated and then recombined. Note that mononuclear cells sedimenting to the bottom of the gradient, although enriched in the relative frequency of T cells, were able to lyse only 1 % of TABLE 1. Cytotoxicity of mononuclear cell subpopulations a Lymphocyte population % Positive cells s RBC rosettes Ig-bearing Cytotoxicity Control 70.4 ± ± ±3.5 Interface 14.5 ± ± ± 6.0 Pellet 93.0 ± ± ± 1.0 a Peripheral blood mononuclear cells from patients with idiopathic colitis were mixed with sheep red blood cells (S-RBC) and AB serum to form S-RBC rosettes. The bulk of rosette-forming cells was separated from non-rosette forming cells by sedimentation over ficollhypaque. Cell fractions at the top (interface) and bottom (pellet) of the ficoll-hypaque as well as cells separated and then recombined (control) were treated with Tris-buffered NH.CI to lyse the S-RBC. Lymphocytes in each fraction were assayed for 1) their ability to reform rosettes with S-RBC, 2) the relative frequency of small round cells positively stained with F1-anti-humai1 Ig, and 3) the ability of cells in each fraction to lyse colonic epithelial cells. Results are expressed as the arithmetic mean ± one SEM for five experiments.

3 Febl'uary1976 INFLAMMATORY BOWEL DISEASE 173 the colonic epithelial cells. Mononuclear cells remaining at the top of the gradient (interface), on the other hand, demonstrated a 5-fold decrease in the relative frequency of cells capable of forming rosettes with S-RBC and a 2-fold increase in the ability of these cells to lyse the target epithelial cells. Hypotonic lysis of the S-RBC in each fraction with Tris-buffered NH 4 CL did not affect the subsequent ability of cells to either reform S-RBC rosettes or to bind Fl. anti-ig to their surface. Thus the relative frequency of T and B cells in the control population was similar to that noted for lymphocytes not treated with the Tris-buffered NH 4 CL (T = 71 ± 6%, B = 20 ± 7%). That the decrease in cytotoxicity noted for T cells sedimenting to the bottom of the gradient was not due to the presence within this fraction of large numbers of nonviable cells was demonstrated by the finding that trypan blue exclusion by mononuclear cells existing in this fraction (84% exclusion) was not drastically different than that noted for either control cell populations (90% exclusion) or cells remaining at the top of the gradient (88% exclusion). Moreover, cells recovered from the pelleted fraction of the gradient demonstrated a brisk response to the T -dependent mitogens phytohemagglutinin and concanavalin A, indicating that a portion of these cells were at least capable of synthesizing new DNA. It is possible that the noted decrease in cytotoxicity for rosette-forming cells sedimenting to the bottom of the gradient was due to enrichment, within this fraction, of a population of cells which could suppress cytotoxic cells also existing within this fraction. Experiments designed to determine this proceeded as follows. Various numbers of T-enriched populations obtained as described above from patients with IBD were added to various numbers of whole PBM from the same patient. These admixtures were then incubated at equivalent cell concentrations with colonic epithelial cells. While the T -enriched cells resulted in 1 % cytotoxicity and the whole mononuclear cell population in 28% cytotoxicity, admixtures containing equal proportions of both cell populations lysed 13% of the target cells. Moreover, cytotoxicity values close to those predicted by the mixing of cytotoxic and noncytotoxic cells were obtained when the relative proportions of T-enriched to whole mononuclear cells was 1:2 and 2:1 (18 and 10% respectively). Thus, we were not able to demonstrate inhibitory influences for cytotoxicity existing within the T -enriched populations. It would appear then that the cytotoxic PBM from patients with IBD is not contained within the majority of rosette-forming or Tpopulationofcells; To determine if the cytotoxic cell was contained within the Ig-bearing or B cell population of lymphocytes, peripheral blood lymphocytes from patients with IBD were incubated in plastic petri dishes coated with antibodies directed against human Ig. Cells not adhering to the dish were gently pipetted off and adherent cells were removed with a rubber policeman. The relative frequency of rosette-forming and Ig-bearing lymphocytes as well as the ability of the separated cell fraction to lyse colonic epithelial cells was then determined (table 2). TABLE 2. Cytotoxicity of adherent and nonadherent cell populations isolated from petri dishes coated with anti l[r Cell % Positive % S-RBC rosettes Ig-bearing Cytotoxicity Control 70.4 ± ± ± 7.4 Nonadherent 75.2 ± ± ± 10.7 Adherent 27.8 ± ± ± 4.9 Results represent the arithmetic mean ± one SE for four experiments. Control refers to cells separated and then recombined. Note that non adherent cells, although demonstrating a relative decrease in the frequency of Ig-bearing lymphocytes, were enriched for cytotoxic cells when compared with populations which were initially separated and then recombined. Alternatively, cell fractions demonstrating a 3-fold increase in their relative frequency of B cells, were relatively depleted of cytotoxicity. Although these experiments were performed in the {>resence of 0.01 % sodium azide to inhibit endocytosis or movement of cell surface receptors, it is possible that the decrease in cytotoxicity noted for cells adhering to the petri dishes might simply be due to alterations in cell surface receptors induced by the anti-ig on the petri dish rather than to the depletion of a cytotoxic cell. Moreover, it has been suggested that decreases in cytotoxicity noted in other systems after the treatment of cells with anti-ig might not be due to removal of Ig-bearing cells, but rather is due to a blocking of receptors involved in cytotoxicity by the release of complexes formed between antibody and cell surface Ig. To determine if the reduced cytotoxicity noted for cells adhering to the anti-ig coated petri dishes resulted from either of these phenomena, the following experiments were performed. 125I-anti-Ig, labeled by the chloramine T method 16 was added to the anti-ig antibody used to coat the petri dishes, and the amount of cell associated radioactivity for nonadherent and adherent populations determined. Various amounts of soluble antibody, using the 125I-anti-Ig as a tracer, were then added to patients' unseparated mononuclear cells for 40 min at 37 C, so that amounts of antibody comparable to that noted for adherent and nonadherent populations were bound to cell surfaces. These cells were then tested for their ability to lyse colonic epithelium. Despite the binding of amounts of antibody which was 2-fold less, similar, or 2-fold greater than that bound to cells adhering to the anti-ig coated petri dishes, the cytotoxicity of these cells (20, 20, 23% respectively) was similar to that noted for. control populations (21 %). This then implies that the decrease in cytotoxicity noted for cells adhering to anti-ig coated petri dishes could not be explained simply by membrane alterations or by the blocking of receptors crucial for cytotoxicity, initiated by the attachment of anti-ig to cell surfaces. It is known that some, but not all, Ig-bearing lymphocytes, T lymphocytes, as well as mononuclear cells not demonstrating classical T or B markers, including macrophages, bear a surface receptor which is capable of combining with the Fc portion of human Ig Although native Ig molecules bind to this receptor, the

4 174 STOBO ETAL. Vol. 70, No.2 strength of the bond is increased by alterations which occur within the Fc region of Ig during either the interaction of the antibody molecule with specific antigen, or by changes induced during heat aggregation of the immunoglobulin molecule. To determine if the mononuclear cells in patients with IBD which are required for lysis of colonic epithelial cells bear this surface Fc receptor, patients' PBM were incubated in petri dishes coated with heat-aggregated IgG for 40 min at 37 C. When compared with cells that were initially separated and then recombined, cells not adhering to the petri dishes manifested a 3-fold decrease in their cytotoxicity whereas cells adherent to the dish were enriched for their cytotoxic ability (table 3). It is unlikely that aggregates sticking to the surface of non adherent populations were responsible for the decreased cytotoxicity noted in this population (table 3). Thus, the cytotoxicity of mononuclear cell populations made by recombining non adherent and adherent populations did not differ significantly from nonseparated cells. Similar experiments performed with petri dishes coated with the Fab fragments of IgG did not result in adherent cell populations which were enriched for cytotoxicity. Finally, to determine the phagocytic properties of the cytotoxic cell, peripheral blood lymphocytes from patients with IBD were incubated at 37 C for 1/2 hr with a solution of iron filings so that cells actively phagocytizing could ingest the iron. These cells were then passed through polyethylene tubing wrapped around an electromagnet and effluent populations tested for their ability to lyse colonic epithelial cells. Despite a 2-fold reduction in the number of phagocytic cells as determined by the ingestion of latex particles by effluent cells, the cytotoxic ability of these cells (30%) was not decreased and indeed demonstrated an increase in cytotoxicity when compared with peripheral blood lymphocytes which had been incubated with iron but not passed through a magnetic field (23%). Discussion In vitro cell-mediated cytotoxicity systems may be delineated by differences in the nature of the effector cell as well as by differences in the requirement for antibody in the cytotoxicity system Thus, in one system, immunization of animals with nucleated allogeneic cells results in a population of mononuclear cells which are subsequently able to lyse, in vitro, the immunogen. 2o The cytotoxic mononuclear cells generated by the immunization appear to be included within the T lymphocyte TABLE 3. Cytotoxicity of adherent and nonadherent cell populations isolated from petri dishes coated with aggregated [ga Cell % Positive % S-RBC rosettes Ig-bearing Cytotoxicity Control 69.5 ± ± ± 9.0 Nonadherent 75.0 ± ± ± 1.1 Adherent 26.0 ± ± ± 8.5 a Results represent the arithmetic mean ± one SE for five different experiments. Control refers to cells separated and then recombined. populations. 2o It is of particular importance to realize that such cell-mediated cytotoxicity may occur inde' pendently of the humoral or antibody response to the alloantigen. Thus, generation of substantial amounts of antibody directed against the target cell is not crucial in mediating the cytolysis. Moreover, cytolysis is specific for the immunizing antigen in that cells from non-immunized animals or cells from animals immunized with antigenically distinct alloantigens do not lyse the target cell. This specificity is presumably mediated via antigen receptors present on the surface of the effector cell. In another cytotoxicity system, termed antibody dependent, cell-mediated cytotoxicity cells from nonimmunized animals are able to lyse, in vitro, certain target cells. However, the target cells must be coated with antibodies directed against their own surface determinants. It has been possible to demonstrate that the cytotoxic cell in this system bears a surface receptor capable of combining with the Fc portion of Ig Presumably it is the interaction between this receptor and the Fc portion of the antibody molecule attached to the target cell which allows close approximation between effector and target and thus initiates cytolysis. Although it can be demonstrated that the mononuclear effector cell is independent of thymic influence,26 the exact ontological derivation of the cytotoxic cell has not been established. This may reflect the heterogeneity of cell types bearing Fc receptors with different cells manifesting different cytotoxic capabilities depending on the nature of the target cell. Recently, it has been suggested that mononuclear cells active in some antibody-dependent, cell-mediated cytotoxicity systems may actually reside within the monocyte-macrophage series These cells lack classical T or B cell markers and thus the term "null" or "K" (killer) has been proposed to denote their ontological-functional relationship.9, 28 In light of this discussion, the data presented in this manuscript can be examined to determine which of the two systems the in vitro lysis of colonic epithelial cells by mononuclear cells from patients with IBD most closely resembles. This has more than theoretical importance, as it may allow certain con{:lusions concerning the in vivo pathogenesis of colonic destruction in these patients. Two points are apparent from the data. Firstly, the cytotoxic cell is not included within the bulk of rosetteforming (T) or Ig-bearing (B) lymphocytes. Indeed, enrichment for cytotoxicity is accompanied by enrichment for a cell population lacking T or B markers. Secondly, cytotoxicity is enriched among cell populations adhering to polystyrene petri dishes coated with aggregated IgG, but not to polystyrene dishes coated with Ig lacking the Fc piece. This indirectly implies that the effector cell bears a surface Fc receptor. Thus, as regards surface receptors present on the effectjr cell, the in vitro lysis of colonic epithelial cells appears to resemble most closely antibody-dependent, cellmediated cytotoxicity. However, no exogenous antibody was added to the assay system. It is unlikely that antibody is already attached to the colonic epithelial cells. Thus, the cells

5 February 1976 INFLAMMATORY BOWEL DISEASE 175 are trypsinized before use, a procedure which proteolytically removes surface-associated Ig. Furthermore, if the epithelial cells were coated with antibody, one would expect that they would be lysed by mononuclear cells from normal individuals. As indicated in Materials and Methods, this is not the case. It is possible that B lymphocytes from patients with IBD are able to generate, during the 4-hr assay, antibody which can sensitize the colonic epithelial cells. If this is true, qualitative differences among B cells must exist in this regard. Thus, cell populations adhering to Ig-coated petri dishes, which contained 66.6% B cells, manifested a 3.5-fold decrease in cytotoxicity when compared with the nonadherent cells containing 13.3% B cells. Antibody then does not appear to be present on the target colonic epithelial cells before their incubation in the assay, nor does it appear that antibody is synthesized by the majority of patients' B cells. If this system does represent antibody-dependent cell-mediated cytotoxicity, then a mechanism must exist for antibody to be present in the system. The following suggests such a mechanism. It has been demonstrated that prior coating of Fc receptor bearing cells with antigen-antibody complexes specifically arms these cells for lysis of target cells bearing the antigen present in the complex. 29, 30 Thus, incubation, in vitro, of human peripheral blood mononuclear cells with complexes of anti-chicken red blood cell-chicken red blood cell antigens, followed by repeated washings, yields mononuclear cells which are specifically cytotoxic in vitro for chicken red blood cells. The armed effector cells are not thymus dependent, and the complexes presumably bind to cells via an Fc receptor. 30 Thus, this arming can be competitively inhibited by heat-aggregated Ig.30. That peripheral blood mononuclear cells from patients with IBD may be similarly armed, in vivo, for the lysis of colonic epithelial cells is suggested by the following. Circulating antibodies with specificities for antigens residing among colonic cells have been detected in the serum of patients with IBD.! Moreover, circulating antigen antibody complexes capable of combining to cell surface Fc receptors have also been detected in a portion of these patients. 3! These data suggest that humoral factors potentially capable of binding to cell surface Fc receptors, thus "arming" these cells for lysis of colonic epithelial cells, do exist in patients with IBD. A more direct demonstration that this may occur is the observation made by Shorter et al. 32 These workers demonstrated that high molecular weight ( > 200,000 mol wt) Ig-containing fractions isolated from the serum of patients with IBD could render normal peripheral blood mononuclear cells cytotoxic for colonic epithelium. Although it was concluded, indirectly, that this represented the attachment of cytophilic IgM to the cells, it is certainly possible that the Ig-containing fractions actually represented complexes of antibodies and colonic antigens, On the basis of these data, we suggest that the in vitro lysis of colonic epithelium by mononuclear cells from patients with IBD represents antibody-dependent, cell-mediated cytotoxicity. These effector cells required for cytolysis bear a surface Fc receptor and cannot be classified, at least by surface receptors for S-RBC and surface Ig determinants, as residing within the bulk of T or B lymphocyte populations. The noted differences in the ability of mononuclear cells from patients with IBD and normal individuals to lyse colonic epithelial cells does not represent any qualitative or quantitative differences in existing Fc receptor bearing cells. Rather, complexes of antibodies and colonic antigens existing only in patients with IBD and not in normal subjects are capable of arming these Fc receptor bearing cells, in vivo, for cytotoxicity. Clearly, much work remains to establish definitely that the above scheme is true. However, this hypothesis does serve to establish a link between humoral and cellular immune mechanisms in the pathogenesis of the colonic inflammation in patients with IBD. Moreover, the hypothesis is directly testable experimentally, and provides an exciting model for possible characterization of antigens involved. Most importantly, because this cytotoxicity was found in patients with both Crohn's disease and ulcerative colitis, it may suggest that these diseases share common pathogenic mechanisms. One last point to be discussed is the specificity of the in vitro cytotoxicity, with regard to the target cell lysed. Previous work has demonstrated that peripheral blood mononuclear cells from patients with IBD contain a material which will lyse target cells other than colonic epithelium. This material may be physically extracted from the cells, or it may be released once the effector cells have been activated by contact with colonic epithelium. 4,5 As shown in Materials and Methods, cytotoxicity in patients with IBD could be elicited only by incubation with colonic epithelium and not by incubation with cells from gastric or duodenal mucosa. Thus, although lysis of colonic epithelium may be triggered by contact with those specific antigens, soluble substances mediating the lysis may lack antigen specificity. Indeed, Granger has demonstrated that cytotoxic cells can be triggered by specific antigens to release mediators which lyse other, antigenically unrelated targets.33 The concept that activation for function may be specific but that soluble materials mediating the function may act without regard for antigen specificity, has recently been demonstrated for cells regulating immunologic responsiveness. 34 The possible in vivo consequences of the release of such "lymphotoxins" in terms of causing destruction of cells in close proximity to colonic epithelium are conjectural. Although the in vitro lysis of colonic epithelium by peripheral blood mononuclear cells from patients with IBD may represent a useful in vitro correlate with disease activity, its exact relationship to pathogenetic mechanisms operative, in vivo, must be interpreted with caution. Whether this in vitro phenomena represents mechanisms initiating colonic destruction, mechanisms by which colonic inflammation is maintained, or merely represents an associated phenomena with no relationship to the disease process is not known. However, if the in vitro cytotoxicity is related to the in vivo disease process, characterization of the cytotoxic cell may have therapeutic relevance. Thus, the ability to deline:lte

6 176 STOBO ET AL. Vol. 70, No.2 functional subpopulations of mononuclear cells by differences in cell surface receptors enables an assessment of the effects of agents such as corticosteroids and other immunosuppressive agents on these subpopulations. In this regard, it has been demonstrated that chronic in vivo administration of either prednisone or azathioprine results in a diminution in the frequency of circulating cells which bear a surface Fc receptor. 35, 36 Further elucidation of the heterogeneity of cytotoxic mononuclear cells which bear a receptor capable of combining with the Fc portion of Ig and their role in the pathogenesis of IBD may thus provide more rationale for therapeutic intervention in this disorder. REFERENCES 1. Perlmann P, Hammarstrom S, Langercrantz R: Immunologic features of idiopathic ulcerative colitis and Crohn's disease. Rendic Gastroenterol 5:17-28, Broberger 0, Perlmann P: Ulcerative colitis. I. 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