Geoffrey S. Nash

Alterations of IgM, IgG, and IgA Synthesis and Secretion by Peripheral Blood and Intestinal Mononuclear Cells from Patients with Ulcerative Colitis and Crohn's Disease

We examined during 12 days of in vitro culture synthesis and secretion of IgM, IgG, and IgA by isolated intestinal mononuclear cells from normal subjects, and from patients with ulcerative colitis, and Crohns disease as well as by peripheral blood mononuclear cells from patients with ulcerative colitis, Crohns disease, and systemic lupus erythematosus. In comparison with normal peripheral blood mononuclear cells, normal intestinal mononuclear cells exhibited moderately increased spontaneous secretion of IgG and IgM and markedly increased spontaneous secretion of IgA. In contrast (when compared with normal intestinal mononuclear cells), Crohns disease and ulcerative colitis intestinal mononuclear cells exhibited decreased spontaneous antibody secretion. Pokeweed mitogen stimulation resulted in only moderate or no increase in antibody secretion. Investigation of tritiated thymidine incorporation revealed completely normal patterns with low spontaneous blast transformation by all intestinal mononuclear cell populations and a marked increase after pokeweed mitogen stimulation. Examination of antibody secretion by peripheral blood mononuclear cells from active, untreated patients with ulcerative colitis, Crohns disease, and systemic lupus erythematosus revealed that all exhibited similar patterns: moderately increased spontaneous synthesis and secretion of IgG and IgM, and markedly increased synthesis and secretion of IgA by unstimulated mononuclear cells over a 12-day period. Stimulation with pokeweed mitogen resulted in little or no increase and, in some instances, suppression of antibody secretion. In all instances, the predominant antibody produced was IgA, which was spontaneously secreted in greater amounts than either IgG or IgM. There are two possible explanations for these results. The first, is that IgA precursor cells are in a highly activated state in normal intestinal lamina propria and because of intestinal inflammation migrate from the intestine into the peripheral blood in increased numbers. The alternative explanation is that a primary mucosal immunodeficiency is present in the intestines of inflammatory bowel disease patients, thus allowing agents to penetrate the intestinal mucosa, and subsequently initiate both a local inflammation and a heightened systemic immune response. In either case, the present studies demonstrate that patients with ulcerative colitis and Crohns disease exhibit major alterations of in vitro synthesis and secretion of immunoglobulins in general and IgA in particular.

Inhibition of Antibody Secretion by 5-Aminosalicylic Acid

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.

Altered patterns of secretion of monomeric IgA and IgA subclass 1 by intestinal mononuclear cells in inflammatory bowel disease.

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.

Alterations in serum immunoglobulin G subclasses in patients with ulcerative colitis and Crohn's 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 Crohns 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 Crohns 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 Crohns 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 Crohns 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.

Antibody secretion by human intestinal mononuclear cells from normal controls and inflammatory bowel disease patients.

Inflammatory bowel disease (IBD) intestinal mononuclear cells (MNC) exhibit decreased spontaneous IgA secretion with an increased percentage of monomeric IgA and IgA subclass 1 in both ulcerative colitis and Crohns disease patients. When compared with control intestinal MNC, a marked increase in spontaneous secretion of IgG is observed from IBD MNC. The greatest increase in spontaneous IgG secretion is seen with ulcerative colitis intestinal MNC, due to the secretion of large amounts of IgG subclass 1. Crohns disease intestinal MNC have increased IgG subclass 2 secretion. Similar differences in IgG subclass concentrations also occur in the sera of active, untreated, IBD patients. Therefore, major alterations occur with regard to spontaneous antibody secretion of IgA and IgG subclasses in IBD. Because intestinal MNC comprise a unique immunologic compartment, it will be important to better understand the regulatory mechanisms, effector capabilities, and inducing antigens involved in intestinal IgA and IgG subclass secretion in IBD.

Human B-cell mitogenic responsiveness to lectins: The requirement for T cells

Abstract The mitogenic potential of normal human peripheral blood lymphocyte subpopulations in response to the plant lectins phytohemagglutin, erythroaggluting phytohemagglutinin, leukoagglutinating phytohemagglutinin, and pokeweed mitogen has been examined. Doubly purified B-, Null, and T-cell fractions were used by themselves and in conjunction with small percentages (1 to 10%) of other subpopulations. Purified human B and Null cells were found to be nonreactive. However, when incubated with as little as 1% T cells, significant mitogenic responsiveness by B and Null cells was observed. This helper effect was apparent with both normal and irradiated T cells. It would thus appear that mitogenic responsiveness of unseparated mononuclear cells is predominantly a function of T cells either as responding cells or as necessary helper cells.

A rapid miniaturized solid-phase radioimmunoassay technique for secreted immunoglobulins, employing microtiter plates, antibody bound to polyacrylamide beads, and filter strip harvesting☆

We have developed a solid-phase radioimmunoassay technique, which allows a large number of samples to be assayed, while minimizing amounts of reagents and technician time. The assay is run in microtiter plates, which results in improved organization, shorter assay set-up time and advantageous miniaturization. On the first day only 3 reagents are required: sample or standard, a 125I-labeled antigen, and an antibody attached to a polyacrylamide bead. After on overnight incubation period, the assay is harvested using a commercial microharvesting apparatus (24 samples/2 min) and placed directly into a gamma counter for counting. In the present study, we have developed the assay to measure secreted IgG, IgM and IgA, and we have compared our results with those of a standard radioimmunoassay technique. This rapid, simple, economical approach should be applicable to the radioimmunoassay of other substances as well.

Altered patterns of secretion of IgA and IgG subclasses by ulcerative colitis and Crohn's disease intestinal mononuclear cells.

Ulcerative colitis (UC) and Crohn’s disease (CD) are inflammatory bowel diseases (IBD) due to unknown etiologic and potentiating factors resulting in immune responses of a chronic inflammatory nature (1). There is an increase in cytoplasmic, surface, and secreted antibodies from IBD intestinal lymphocytes, due mainly to enhanced expression and production of IgG (2–4). In previous studies (5,6) we have found that immunoglobulin secretion patterns by peripheral blood and intestinal mononuclear cells (MNC) from inflammatory bowel disease patients is altered. IBD peripheral blood MNC reveal a markedly increased spontaneous secretion of IgA which is partially suppressed by PWM (5,6). Intestinal MNC from control specimens also spontaneously secrete large amounts of IgA, which is suppressed by PWM (5,6). In IBD patients, intestinal MNC exhibit decreased spontaneous IgA secretion, but have increased IgG secretion compared with control intestinal MNC (5–7). The changes in antibody secretion observed in IBD could be due to: (a) preferential proliferation of subpopulations of cells due to immunoregulatory alteration; (b) switching of the isotype and/or subclass of antibody secreted by the lymphocytes themselves; or (c) changes in the homing and trafficking patterns of the lymphocytes due to the inflammatory process.

Activation of human lamina propria mononuclear cells in inflammatory bowel disease

The state of activation of human lamina propria mononuclear cells (LPMnc) obtained from either organ transplant donors or from operation specimens from inflammatory bowel disease (IBD) patients were studied by three-color fluorescence activated cell sorting (FACS). Freshly isolated LPMnc from normal donors as well as from IBD patients were autofluoresent. The autofluorescence could be subtracted from the FACS data on a cell-by-cell basis by a mathematical post processing step. Normal LPMnc are in vivo activated and express increased levels of 4F2-antigen, transferrin and interleukin-2 receptor. The number of lymphocytes, which express these early activation antigens was markedly increased in both Ulcerative Colitis (U.C.) and Crohn’s disease (C.D.). This underlines the hypothesis of a major upregulation of the mucosal immune system due to the disease process. B cells in particular were activated, suggesting they may be of particular importance in the disease process of IBD.

Human rib bone marrow mononuclear cells spontaneously synthesize and secrete IgE in vitro.

We have examined spontaneous secretion of IgE by human rib bone marrow mononuclear cells (MNC). Bone marrow MNC from nine out of 12 rib specimens synthesized and secreted substantial amounts of IgE during 14 days of in vitro culture. The 14‐day supernatants from these bone marrow MNC contained a mean of 2589 pg/ml of IgR (n= 12) with a maximum production of 1540K pg/ml of IgE compared with small amounts of IgE 80–200 pg/ml) produced by similarly cultured normal and inflammatory bowel disease intestinal lamina propria MNC. Using two rib specimens, time‐course studies revealed spontaneous secretion of IgE to be minimal during the first 2 days of culture (152 pg/ml), followed by a steady increase between days 4 (517 pg/ml) and 14 (3588 pg/ml). The addition of pokeweed mitogen resulted in 72% suppression of spontaneous IgE production by bone marrow MNC. The bone marrow MNC isolated from the ribs consisted of 22% Leu 12+ (B) cells of which 3.2% were surface IgE positive. Staining for cytoplasmic immunoglobulin revealed 1% of the bone marrow MNC to be cytoplasmic IgE+. The presence of IgE‐bearing and IgE‐secreting MNC in human bone marrow is consistent with the observation that allergen‐specific IgE‐mediated hypersensitivity is adoptively transferred by human bone marrow transplantation and demonstrates the usefulness of human bone marrow MNC for examination of IgE secretory and regulatory events.