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Background: Monocyte-chemoattractant protein 1 (MCP-1) activates macrophages and increases the migration of monocytes into tissue during inflammation. It was hypothesized that MCP-1 expression is involved in intestinal inflammation., Methods: MCP-1 protein was detected by immunohistochemistry and immunoprecipitation. Biological activity of MCP-1 was assessed using a chemotactic assay. MCP-1 messenger RNA (mRNA) levels were measured by quantitative reverse-transcription polymerase chain reaction., Results: In normal mucosa, MCP-1 was predominantly present in surface epithelium. In contrast, inflamed mucosa from patients with ulcerative colitis or Crohn's disease contained multiple cells immunoreactive for MCP-1, including spindle cells, mononuclear cells, and endothelial cells. Furthermore, MCP-1 mRNA expression was markedly increased in inflamed intestinal biopsy specimens from patients with inflammatory bowel disease. MCP-1 was detected in isolated intestinal epithelial cells and in conditioned media from Caco-2 cells. Caco-2 cell-conditioned media stimulated monocyte chemotaxis activity that was inhibited by anti-MCP-1 antibodies. Constituitive MCP-1 mRNA levels in Caco-2 cells were up-regulated by interleukin 1 beta and down-regulated by dexamethasone., Conclusions: In addition to lamina propria macrophages, endothelial cells, and spindle cells, intestinal epithelial cells are able to produce MCP-1. MCP-1 is expressed constitutively in the intestinal colonic mucosa and is up-regulated during inflammation.

Background: Inflammatory bowel disease occurs in regions of the intestine characterized by a bowel content high in bacteria. Intestinal bacteria synthesize cell wall products such as lipopolysaccharide; when normal monocytes or macrophages come in contact with these products, they can be primed to release a number of inflammatory mediators. Mediators such as toxic oxygen metabolites released as part of the respiratory burst may contribute to inflammatory tissue damage. The aim of this study was to determine if monocytes from patients with Crohn's disease are primed by lipopolysaccharide for a greater respiratory burst., Methods: The generation of superoxide anion was measured by superoxide dismutase inhibitable reduction of ferricytochrome c., Results: Freshly isolated monocytes from active untreated Crohn's disease patients (n = 8) showed enhanced stimulated release of superoxide anion when compared with normal monocytes (n = 15; 3.80 +/- 0.12 vs. 1.02 +/- 0.06 nmol/5 min; P < 0.001). We tested the hypothesis that the monocyte priming factor in Crohn's disease serum may be lipopolysaccharide by showing that Crohn's disease serum lost its ability to prime normal monocytes after lipopolysaccharide was removed (0.25 +/- 0.25 nmol/5 min, P < 0.001)., Conclusions: These studies indicate that bacterial cell wall products may be important proinflammatory molecules involved in the initiation and/or perpetuation of Crohn's disease.

Normal human lamina propria lymphocytes are in a heightened state of activation compared with peripheral blood with regard to cell-surface activation antigen expression (transferrin receptor, interleukin-2 receptor, 4F2) and the increased spontaneous secretion of immunoglobulins in vitro. This study evaluates the cell-surface expression of activation-associated antigens in different subpopulations of isolated colonic lamina propria mononuclear cells in inflammatory bowel disease. In pilot studies using three-color flow cytometry, autofluorescence was observed that was emitted by unstained lamina propria mononuclear cells, which interfered with both the sensitivity and the specificity of the analyses. Because a major portion of the intestinal lymphocyte populations of interest were autofluorescent, a method to remove autofluorescence signals was developed by designing a computer program for the subtraction of autofluorescence from the emissions of each individual cell. This technique increases both the sensitivity and specificity of flow-cytometric analyses of intestinal lamina propria mononuclear cells. Using fluorescence-activated cell-sorter analyses with subtraction of autofluorescence on a single-cell basis, increased expression of lymphocyte activation antigens (interleukin-2 receptor, transferrin receptor, 4F2) was found on the cell surface of isolated intestinal B cells, T cells, CD4+ T cells, and CD8+ T cells in both Crohn's disease and ulcerative colitis. Therefore, markedly increased intestinal lymphocyte activation is a major immunological alteration in inflammatory bowel disease and includes all lymphocyte subpopulations investigated in this study. In addition, 5-aminosalicylic acid, which is used for the treatment of intestinal inflammation in inflammatory bowel disease, inhibits the expression of cell-surface activation antigens on mitogen-activated peripheral blood lymphocytes in a dose-dependent manner. These observations suggest that lymphocyte activation may play an important role in underlying immune processes that lead to chronicity and perpetuation of inflammatory bowel disease and may implicate an additional mechanism for the therapeutic action of 5-aminosalicylic acid.

Intestinal mononuclear cells (MNC) from patients with inflammatory bowel disease (IBD) demonstrated altered patterns of spontaneous secretion of immunoglobulin A (IgA). Control intestinal MNC had markedly high spontaneous secretion of IgA compared to IBD intestinal MNC. Control intestinal MNC secreted predominantly dimeric IgA (only 31% monomeric IgA), whereas IBD intestinal MNC secreted increased amounts (43%-53%) of monomeric IgA. Control intestinal MNC secreted 61% IgA subclass 1 (IgA1), whereas IBD intestinal MNC secreted 71%-74% IgA1. Intestinal MNC from involved portions of resected specimens secreted more of both monomeric IgA and IgA1 than MNC from uninvolved areas of the same bowel. Therefore, intestinal MNC from involved IBD intestinal specimens secrete less total IgA but high percentages of monomeric IgA and IgA1 compared to control intestinal MNC. This could be caused by increased homing of monomeric IgA- and IgA1-producing cells into involved intestine, or the in situ proliferation of monomeric IgA and IgA1 precursor cells resulting from immunoregulatory alterations. These observations may represent the normal mucosal IgA immune response to infectious agents or inducing factors of either a primary or a secondary nature.

These studies were designed to determine the ability of antihepatocyte antibodies from patients with chronic active liver disease to induce killing of rabbit hepatocytes by normal lymphocytes. Normal subjects and patients with chronic persistent hepatitis or chronic active liver disease served as sources of sera, and normal human peripheral lymphocytes and their subsets (T-enriched, K-enriched, and B) served as effectors. Only sera from patients with chronic active liver disease possessed membrane-reactive IgG directed against the surface of rabbit or human hepatocytes. After pretreatment of rabbit hepatocytes with sera from patients with chronic active liver disease, marked cytotoxicity mediated by normal lymphocytes was observed. K-enriched cells mediated the antibody-dependent cellular cytotoxicity, whereas T-enriched cells and B-cells did not. Heat-aggregated human IgG blocked the antibody-dependent cellular cytotoxicity. These data suggest that sera from patients with chronic active liver disease contain IgG antihepatocyte antibodies, which are capable of inducing normal K-cells to kill rabbit hepatocytes in vitro.

To examine the possible contribution of cellular immunoregulatory mechanisms to the pathogenesis and progression of alcoholic liver diseases, suppressor T-cell function was evaluated in patients with severe active or inactive alcoholic liver disease and compared with normal control subjects. Suppressor T-cell activity after in vitro induction by concanavalin A or other T cell-mediated immune reactions was then assessed. T-cell interactions studied included the proliferative responses of both autologous or allogeneic responding peripheral blood mononuclear cells to T-cell mitogens and to allogeneic cells. No significant differences were found in the ability to induce suppressor T cells between controls and patients with inactive alcoholic liver disease (p < 0.05). In contrast, T cells from patients with severe active alcoholic liver disease failed to develop suppressor cell activity (p < 0.001) after concanavalin A stimulation; restoration of suppressor cell function was found after these patients improved clinically. Adherent cells (monocyte-macrophages) from patients with active alcoholic liver diseases exhibited normal support of concanavalin A-induced blastogenesis and suppressor T-cell generation. This immunoregulatory cell abnormality could be important in the pathogenesis and/or progression of active alcoholic liver disease.

We have examined the T-cell functional capabilities of human intestinal mononuclear cells isolated from surgically obtained normal and inflammatory bowel disease intestinal specimens. Intestinal mononuclear cells have T cells present which respond to the mitogenic lectins E-PHA, Con A, and PWM and to foreign cell-surface antigens in the allogeneic-mixed leukocyte reaction. Intestinal mononuclear cells from ulcerative colitis patients exhibit a significantly decreased responsiveness in comparison to normal intestinal mononuclear cells with regard to both mitogenesis and the allogeneic-mixed leukocyte reaction; however, these defects may be secondary to the severity of the disease process that led to intestinal resection or the therapy which patients had received. Although both normal and inflammatory bowel disease intestinal mononuclear cells exhibit responsiveness in the allogeneic-mixed leukocyte reaction, thus indicating recognition of and sensitization by foreign cell-surface determinants, intestinal mononuclear cells do not subsequently kill the sensitizing cells in cell-mediated lympholysis. Therefore, the subclass of T cells which mediates cell-mediated lympholysis may either be absent from intestinal mononuclear cells or nonfunctional, while the subclass of T cells which responds in the allogeneic-mixed leukocyte reaction is both present and functional. This observation adds to the evidence of major functional differences between intestinal and peripheral blood mononuclear cells. Therefore, it will be necessary to better understand the factors regulating the effector capabilities of intestinal mononuclear cells before delineation of immunopathologic events in these tissues.

We have examined the T-cell functional capabilities of peripheral blood mononuclear cells from 32 active or inactive patients with ulcerative colitis and Crohn's disease by examining blastogenic responsiveness in the allogeneic and autologous mixed leukocyte reaction and cytotoxic capabilities in cell-mediated lympholysis. Severely ill, malnourished, or preoperative patients, or patients being treated with greater than 10 mg of prednisone per day were excluded from study. Studies using the allogeneic mixed leukocyte reaction revealed no decreased responsiveness by inflammatory bowel disease peripheral blood cells. In cell-mediated lympholysis, healthy control peripheral blood mononuclear cells exhibited 17% cytotoxicity, and there was no significant difference in killing exhibited by patients with inflammatory bowel disease. In study of the autologous mixed leukocyte reaction, cells were separated using techniques that included exposure to xenoantigens (fetal calf serum and sheep red blood cells), and peripheral blood cells from inflammatory bowel disease patients did not exhibit major defects in responsiveness. Therefore, in these experiments, we have detected no significant depression in three major T-cell functions by peripheral blood cells in moderately ill patients with ulcerative colitis or Crohn's disease. We conclude that defects in these capabilities may not be primarily causative in the immunopathogenesis of inflammatory bowel disease or the increased incidence of cancer seen in ulcerative colitis. Instead, when defects in these T-cell functions are observed, they may be influenced by the techniques used or may represent secondary in vitro immunologic events related to disease severity, inflammation, medications, or other causes that are not specific for inflammatory bowel disease.

We have examined the concentration of immunoglobulin G (IgG) subclass antibodies in the sera of 27 patients with ulcerative colitis and 21 patients with Crohn's disease as well as in 11 normal controls and 11 patients with systemic lupus erythematosus. In comparison with a control mean serum IgG1 concentration of 5173 micrograms/ml, patients with ulcerative colitis exhibited a significantly increased mean serum concentration of 7924 micrograms/ml (p less than 0.05), whereas patients with Crohn's disease had a near normal mean serum IgG1 level of 5898 micrograms/ml. In contrast, control sera had a mean IgG2 level of 2477 micrograms/ml and ulcerative colitis sera had a similar IgG2 level of 2269 micrograms/ml, whereas Crohn's disease sera had a significantly increased mean IgG2 level of 5111 micrograms/ml (p less than 0.05). Patients with systemic lupus erythematosus, like those with ulcerative colitis, had a markedly elevated serum IgG1 level of 15,594 micrograms/ml (p less than 0.001) without a significantly increased IgG2 serum level (3271 micrograms/ml). Neither ulcerative colitis nor Crohn's disease sera exhibited altered levels of IgG3 or IgG4. These data show that alterations in IgG subclass concentrations occur in the sera of patients with active, untreated inflammatory bowel disease, similar to the previously noted changes in the IgG subclasses secreted by lymphocytes from involved inflammatory bowel disease intestinal specimens.

Intestinal mononuclear cells are poor mediators of spontaneous and antibody-dependent cellular cytotoxicity. In this study, we found that both interferon and mitogenic lectins were able to induce increased levels of cell-mediated cytotoxicity by intestinal mononuclear cells. Intestinal mononuclear cells from patients with inflammatory bowel disease exhibited hyporesponsiveness to cytotoxic activation by interferon or lectins compared with control intestinal mononuclear cells. Peripheral blood mononuclear cells from patients with Crohn's disease exhibited deficient spontaneous and antibody-dependent cytotoxicity that could be partially reversed by interferon or mitogenic lectins. These studies demonstrate that exogenous agents or endogenous factors can induce deficient intestinal and peripheral blood cytotoxic effector cells from inflammatory bowel disease patients to become active. In comparison with control cells, however, intestinal and peripheral blood mononuclear cells from inflammatory bowel disease patients are not only deficient in cytotoxic capabilities but also are hyporesponsive to interferon and lectin activation.

We have examined the effects of sulfasalazine and its metabolites sulfapyridine and 5-aminosalicylic acid on antibody secretion by normal peripheral blood and intestinal mononuclear cells. Sulfasalazine and 5-aminosalicylic acid both inhibited pokeweed mitogen-stimulated secretion of immunoglobulins (Igs) A, G, and M by peripheral blood mononuclear cells in a dose-dependent manner, whereas sulfapyridine had little effect. Sulfasalazine and 5-aminosalicylic acid also inhibited spontaneous secretion of IgA by intestinal mononuclear cells, but sulfapyridine did not. Sulfasalazine inhibited pokeweed mitogen-stimulated lymphocyte proliferation, while 5-aminosalicylic acid and sulfapyridine exhibited minimal inhibition. Sulfasalazine was toxic for peripheral blood mononuclear cells, whereas 5-aminosalicylic acid and sulfapyridine were not toxic. Thus, the inhibition of antibody secretion by sulfasalazine was due to direct toxicity. On the other hand, 5-aminosalicylic acid, the therapeutically active component of sulfasalazine, was neither toxic nor antiproliferative, and appeared to exert its effects on metabolic pathways directly related to antibody synthesis. The calculated ID50 values of 5-aminosalicylic acid for antibody secretion were 1.35 mM for IgA and 1.05 mM for IgG, concentrations that are achieved in the colons of treated individuals. Indomethacin did not inhibit antibody secretion at pharmacologically relevant concentrations. 5-Aminosalicylic acid mediated inhibition of antibody secretion may play a role in inflammatory bowel disease by stopping antibody-mediated memory events involved in the induction or perpetuation of the disease process.