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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.

We previously described the presence of an inhibitory protein contained in the 20 to 40% (NH4)2SO4 precipitable fraction of FCS that down-regulates expression of mannose receptors on bone marrow-derived macrophages. We now identify aggregated bovine IgG as the main inhibitory component. Heat-aggregated bovine IgG was capable of down-regulating expression of the macrophage mannose receptor in a dose-dependent manner without inducing changes in ligand affinity whereas neither F(ab')2 fragments nor nonaggregated IgG displayed any inhibitory effect. Depleting of IgG from heat inactivated FCS by protein G affinity chromatography completely removes the inhibitory activity. Moreover, readdition of the Ig eluate from the protein G chromatography column restored inhibition in a dose-dependent manner. Macrophages were able to clear exogenously added aggregated bovine IgG, thus leading to loss of inhibitory activity in macrophage-conditioned media as compared to sham-conditioned media containing aggregated IgG. These results indicate that aggregated IgG down-regulates mannose receptor expression by macrophage activation via interaction with Fc-gamma R.

The macrophage mannose receptor, a 172-kDa lineage-specific glycoprotein, partakes in nonopsonin-mediated phagocytosis by recognition of terminal mannose residues on targeted particles. Because appearance of the receptor progresses with monocyte/macrophage differentiation, its expression is indicative of the maturational state of the cell. Monomeric IgG2a and IgG2b up-regulate mannose-receptor surface expression and biosynthesis by murine bone-marrow macrophage precursors as much as 7- to 12-fold in a dose-dependent manner. IgG2a accelerates macrophage mannose-receptor expression by several days during in vitro bone-marrow differentiation; however, treated and control cells ultimately express equivalent levels of receptor. Moreover, the effect is independent of cell cycle or ambient levels of colony-stimulating factor 1. The coinduction of another maturation-dependent lineage-specific antigen, F4/80, and the fact that macrophage precursors respond to IgG2a only within the first day of culture, indicate that the targeted cell is an early myelomonocytic precursor, responsive only during a short, early developmental window. The effect is specific for immunoglobulin molecules of the IgG2a and IgG2b subclasses and probably involves an Fc gamma-receptor signal-transduction pathway but not macrophage priming or activation. Most importantly, a paracrine mechanism of immunoglobulin-mediated bone-marrow macrophage differentiation is suggested by experiments in which basal levels of mannose-receptor expression are reduced by continual removal of B-cell-generated IgG from marrow cultures. Thus, IgG2a and IgG2b prompt mannose-receptor synthesis and bone-marrow macrophage differentiation and may, therefore, play a role in the regulation of macrophage differentiation in host defense.

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 IgE (n = 12) with a maximum production of 15,408 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% Leu12+ (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.

Reactive oxygen metabolites have been implicated as important mediators of inflammation-induced intestinal injury associated with ischemia (and reperfusion), radiation, and inflammatory bowel disease. Because the colonic mucosa may be subjected to significant oxidant stress during times of acute and chronic inflammation, knowledge of the oxidant defense mechanisms in the colon is of biologic and potential clinical importance. Therefore, the objective of this study was to quantify the specific activities of superoxide dismutase (SOD), catalase, and GSH peroxidase in the normal human colon. We found low, but significant, amounts of all three enzymes in the mucosa, submucosa, and muscularis/serosa of the human colon. However, the mucosal, levels of SOD (3.6 +/- 0.3 units/mg protein), catalase (11 +/- 3 units/mg), and GSH peroxidase (15.2 +/- 0.8 mU/mg) represented only 8%, 4%, and 40%, respectively, of those values determined for human liver. Colonic epithelial cells derived from mucosal biopsies exhibited significantly higher specific activities for SOD (12 +/- 0.5 units/mg) and catalase (26 +/- 6 units/mg) when compared to whole mucosa, suggesting most of the mucosal activity was associated with the epithelial cells and not the lamina propria. In a comparative study, we found that a human colonic carcinoma cell line (CaCo-2) contained significantly lower SOD (6 +/- 0.5 units/mg) and catalase (6 +/- 0.6 units/mg) activities when compared to colonic epithelial cells. Taken together, our data suggest that: (1) the colonic mucosa is relatively deficient in antioxidant enzymes when compared to liver, and (2) most of the protective enzyme activity is localized within the epithelium and not the mucosal interstitium.

We previously observed that milk-derived bovine IgG, but not serum-derived bovine IgG, strongly inhibits antibody secretion by pokeweed mitogen (PWM)-stimulated human peripheral blood mononuclear cells (PBMC). Bovine milk contains a greater percentage of IgG1 (90%) than does bovine serum (53%). To determine whether bovine IgG subclasses have different functional capabilities, we have examined the effects of bovine IgG1 and IgG2 subclasses upon not only antibody secretion but also mitogenesis by human PBMC. Both bovine IgG subclasses markedly inhibited PWM-stimulated mitogenesis. However, only bovine IgG1, and not IgG2, inhibited antibody secretion during a 14-day in vitro culture period. Also, antibody secretion was inhibited following a 24-hr preincubation of human PBMC with bovine IgG1, but not with IgG2. To determine whether these differences corresponded to specificities of human Fc gamma receptors on subsets of mononuclear cells, fluorescence-activated cell sorter (FACS) analyses were performed. Both bovine IgG subclasses bound to human monocytes. However, only bovine IgG1 bound to human B cells, and bovine IgG1 bound more avidly to human B cells than did human IgG. One model to explain these findings is that inhibition of mitogenesis may be due to the binding of both bovine IgG1 and IgG2 subclasses to monocytes; whereas subclass-specific inhibition of antibody secretion may result from the selective binding of bovine IgG1, but not bovine IgG2, to B cells. The observation that bovine IgG1 has a greater avidity for human B lymphocyte Fc receptors than human IgG may have important implications for future studies of Fc gamma receptors on human leucocytes.

In the present experiments, we have observed that cells separated using techniques that provide no xenoantigen exposure to stimulator cells do not participate in the autologous MLR. However, exposure of separated cells in general and stimulator cells in particular to fetal calf serum does lead to autologous MLR. These data confirm the previous observations of Huber et al. that the human autologous MLR is primarily due to xenoproteins absorbed to stimulating cells during the separation procedures and indicate that a reassessment of human autologous MLR studies will be needed.

Peripheral leukocytes from 16 Thai children with dengue hemorrhagic fever were examined to determine the leukocyte composition on the day of presentation and on convalescent days 15 and 30. Mononuclear cells were isolated each time, and the concentrations of T, B, Fc receptor-bearing, and "null" cells were determined. On the day of hospitalization, in comparison to convalescent values, there was a significant increase in total lymphocytes, primarily due to concentrations of atypical lymphocytes. There was a significant loss of T cells with an increase in non-T, non-B, non-Fc receptor-bearing null cells. There were no changes in the concentrations of monocytes, B cells, or Fc receptor-bearing cells when acute and convalescent values were compared. During the convalescent period, a progressive increase in eosinophils was noted. Also, on day 15 but not on day 30 of the convalescent period, an increase was observed in the total leukocyte number due to an increase in granulocytes. There results indicate that in Thai children with dengue hemorrhagic fever, there are major shifts within several component cell subpopulations of the immune system.

Con A-pretreated mononuclear (MNC) cells from Thai adults with naturally acquired P. falciparum or P. vivax malaria were significantly less effective in suppressing the responsiveness of autologous or normal allogeneic responder cells to mitogenic lectins or allogenic stimulator cells than pretreated cells from healthy donors. Serial studies of three patients demonstrated that reduced suppressor cell activity was present early in malaria infection but returned to normal soon after treatment. These studies demonstrate that the loss of T cells previously observed in patients with malaria, in part may functionally represent a loss of suppressor T cells.

In cooperation with human heat-inactivated antisera from adults immunized with group C meningococcal polysaccharide, normal human peripheral blood mononuclear cells significantly decreased the viability of group C meningococci (Mgc) in vitro. K lymphocytes (Null cells) and monocytes, (but not T or B lymphocytes) were capable of effecting antibody-dependent cell-mediated (ADC) antibacterial activity in this system. The degree to which meningococcal viability was decreased was a function of the length of the test incubation, the concentration of effector cells, and the amount of antiserum used in the assay. When specific antibodies directed against Mgc were adsorbed from the antiserum, cell-mediated antibacterial activity was abolished. ADC antibacterial activity was also abrogated by performing the assay at 4 degrees C or by heating effector cells to 46 degrees C for 15 min before the assay, Similarities between the ADC antibacterial system and previously described ADCC assays are discussed. The data suggest the K cells (as well as monocytes) may play a role in host immune defense against pathogenic bacteria.

Human cloned 35S-labeled NK cells were disrupted by nitrogen cavitation, and their secretory granules were obtained by filtration through 5-micron and 3-micron membrane filters followed by Percoll density-gradient centrifugation. These granule preparations, which contained 35S-labeled chondroitin sulfate A proteoglycans, were sonicated and were analyzed for carboxypeptidase activity and tryptic serine esterase activity. A carboxypeptidase activity that digested angiotensin I to des-Leu-angiotensin I, Ile-His-Pro-Phe to Ile-His-Pro and Phe, and hippuryl-L-phenylalanine to hippuric acid and Phe was detected in the granules of these NK cells. As determined by cleavage of the tetrapeptide, the pH optimum of the carboxypeptidase was 7.0. As assessed by the cleavage of N-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLTe), the granule preparations also contained a serine esterase with trypsin-like specificity that had a pH optimum of 8.5. When the isolated secretory granules were disrupted and chromatographed on columns of Sepharose CL-2B in PBS, greater than 60% of the BLTe serine esterase activity and essentially all of the carboxypeptidase activity filtered as a macromolecular complex with approximately 8% of the 35S-labeled proteoglycans. Whereas treatment with 4 M urea or nonionic detergent failed to disrupt the macromolecular complex, the serine esterase activity was dissociated from the macromolecular complex in the presence of 3 M NaCl, demonstrating an ionic interaction with the proteoglycans. No difference was observed in the disaccharide composition of the chondroitin sulfate glycosaminoglycans of the 35S-labeled proteoglycans that were complexed with the enzymes as compared to those that were not complexed. These studies indicate that the secretory granules of human NK cells contain serine esterase activity and carboxypeptidase activity, both of which have neutral pH optima, and both of which are bound to protease-resistant chondroitin sulfate proteoglycans.

The Sézary syndrome is a frequently lethal disease characterized by circulating malignant cells of thymus-derived (T)-cell origin. The capacity of circulating malignant lymphocytes from patients with this syndrome to synthesize immunoglobulins and to function as helper or suppressor cells regulating immunoglobulin synthesis by bone marrow-derived (B) lymphocytes was determined. Peripheral blood lymphocytes from normal individuals had geometric mean immunoglobulin synthetic rates of 4,910 ng for IgM, 1,270 ng for IgA, and 1,625 ng for IgG per 2 X 10(6) cells in culture with pokeweed mitogen for 7 days. Purified normal B cells had geometric mean synthetic rates of 198 ng for IgM, 145 ng for IgA, and 102 ng for IgG. Leukemic cells from patients with the Sézary syndrome produced essentially no immunoglobulins. Adding normal T cells to normal B cells restored their immunoglobin producing capacity. Leukemic cells from four of five patients tested had a similar capacity to help immunoglobulin synthesis by purified normal B cells. Additionally, Sézary cells from one patient studied induced a nearly 10-fold increase in IgA synthesis by lymphocytes from a child with ataxia telangiectasia and selective IgA deficiency. Furthermore, these Sézary cells induced more than a 500-fold increase in IgG and IgA synthesis by lymphocytes from a child with Nezelof's syndrome. When Sézary cells were added to normal unfractionated lymphocytes, they did not suppress immunoglobulin biosynthesis. In addition, unlike the situation observed when large numbers of normal T cells were added to purified B cells, there was no depression of immunoglobulin synthesis at very high malignant T-cell to B-cell ratios. These data support the view that Sézary T cells do not express suppressor cell activity. The results presented in this paper suggest that neoplastic lymphocytes from the majority of patients with the Sézary syndrome originate from a subset of T cells programmed exclusively for helper-like interactions with B cells in their production of immunoglobulin molecules.

The T11 (CD2) antigen has been found to be an alternate pathway for antigen-independent activation of resting T cells. T11 triggering also results in activation of NK cells and enhancement of their cytolytic function. The present studies were carried out to further define the mechanisms whereby cytotoxicity is enhanced after T11 activation. A series of clonal human NK cell lines were analyzed after incubation with monoclonal anti-T112 and anti-T113 antibodies specific for different epitopes of the CD2 protein. Anti-T112/3 triggering resulted in increased cytotoxicity against a variety of target cells. Similar results were obtained with F(ab')2 fragments of anti-T112/3, indicating that this effect was not mediated through binding of FcR. The induction of cytotoxicity was found to be associated with increased formation of effector cell-target cell conjugates and with release of secretory granule-localized 35S-labeled proteoglycans. Both enhanced conjugate formation and cytotoxicity could be blocked by anti-lymphocyte function-associated antigen (LFA-1) mAb. Ultrastructural analysis of NK cells after T11 activation demonstrated increased adherence of effector cells to targets and other NK cells as well as a directional reorientation of cytoplasm and intracellular granules toward the area of contact between cells. Discharge of granules occurred into pockets bounded by closely apposed plasma membranes. In the presence of anti-LFA-1 and anti-T112/3, the close apposition and formation of pockets between effector cells and target cells did not occur but the cells exocytosed their intracellular granules. T11 activation of NK cloned cells also resulted in the formation of the homotypic conjugates and autocytotoxicity. As seen with resistant allogeneic targets, autocytotoxicity was mediated by F(ab')2 fragments of T112/3 antibodies and could be blocked by anti-LFA-1 antibody. Ultrastructural analysis of NK cloned cells after T11 activation confirmed the presence of homotypic conjugates with reorientation of effector cells toward one another and discharge of cytolytic granules into pockets formed between NK cloned cells. Taken together, these results indicate that T11-induced cytolytic function of NK cells is, in part, mediated through increased binding of effector cells and targets and that enhanced conjugate formation is at least in part mediated by the LFA-1 antigen. In addition, T11 activation results in the triggering of the cytolytic mechanism of NK cells and the exocytosis of cytolytic granules and their constituents.

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.

We have previously observed that Thai adults who are infected with malaria have a loss of peripheral blood T cells, and that patient sera contain lymphocytotoxic antibodies. In the present study, we examined peripheral blood mononuclear cells from Thai adults naturally infected with Plasmodium falciparum and Plasmodium vivax for the capacity to undergo blastogenesis in response to phytohemagglutinin, concanavalin A, pokeweed mitogen, and allogeneic cell surface antigens in a one-way mixed leukocyte reaction. In addition, sera from actively infected patients were examined with regard to suppressive capabilities toward normal lymphocyte blastogenesis by using the same assays. We found that patient mononuclear cells exhibited normal reactivity to phytohemagglutinin, concanavalin A, and pokeweed mitogen when compared with controls. However, peripheral blood mononuclear cells from patients had a decreased stimulatory capacity in the allogeneic mixed leukocyte reaction, and P. vivax, but not P. falciparum, lymphocytes exhibited decreased responsiveness in the mixed leukocyte reaction. Furthermore, sera from patients with active malaria induced decreased responsiveness by normal mononuclear cells to phytohemagglutinin and concanavalin A, but not pokeweed mitogen; pooled P. falciparum sera caused decreased responsiveness to allogeneic cell surface antigens in the mixed leukocyte reaction. These studies indicate that despite the lost of circulating T cells during the course of infection with malaria, blastogenic responsiveness remains intact, and that sera from patients with malaria are capable of exerting negative immunoregulatory effects.

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.

Bovine milk IgG markedly inhibits the pokeweed mitogen (PWM)-induced secretion of immunoglobulins from human peripheral blood mononuclear cells. Heat-aggregated bovine milk IgG is even more inhibitory, demonstrating significant inhibition when levels as low as 5-9 micrograms/ml are continuously present in the in vitro 14-day culture system. However, bovine serum IgG, regardless of its state of aggregation, and control proteins have little effect on PWM-induced secretion of human IgG, IgA, and IgM. In a similar fashion, goat milk IgG, especially when aggregated, inhibits human antibody secretion whereas goat serum IgG does not. Inhibition appears to be mediated by Fc gamma receptors since F(ab')2 fragments of milk-derived bovine IgG do not inhibit PWM-induced antibody secretion. The continuous presence of bovine milk IgG is not essential since preincubation of milk IgG with PWM and human mononuclear cells for 24 hr also results in inhibition of human immunoglobulin secretion. In examining potential mechanisms of inhibition, it was found that bovine milk IgG, bovine serum IgG, and another chitin-containing protein, bovine thyroglobulin, each caused a small and equal inhibition of the binding of 125I-labeled PWM to human mononuclear cells, yet only the milk IgG inhibited antibody production. These studies raise the question of whether bovine milk IgG might modulate the human immune system in vivo.

The studies presented herein have evaluated both the specificity and cellular basis of cell-mediated lympholysis (CML) in man. An efficient and quantitative 51Cr release assay was utilized to study the role of highly purified human T and B cells in CML. After in vitro sensitization human T cells develop the capacity to kill specifically allogeneic cells to which they were sensitized. In contrast, B cells were neither triggered to proliferate nor activated to kill allogeneic targets. B cells were not activated to kill even when sensitized in the presence of potentially "helper" T cells, nor did they block T cells from killing during the effector phase. Cell-free supernatants taken from active in vitro sensitization cultures were not lympholytic and did not modulate T cell killing. Hence, these studies show that both the afferent and efferent phases of human CML are T cell functions.

The effects of single oral doses of 10, 15, or 30 mg of prednisone on circulating mononuclear cells, autologous MLR, mitogen responses, and allogeneic MLR were studied in healthy volunteers. Doses as low as 10 mg were immunosuppressive, causing diminution of circulating T cells and monocytes, and significant reduction in autologous but not allogeneic MLR responses. These effects were maximal 6 hr after drug administration and gone by 24 hr. Autologous MLR responses were particularly sensitive to the effects of prednisone being significantly and consistently suppressed 2 hr after drug administration, before significant cell redistribution had occurred. Macrophage-enriched stimulating cells were more easily suppressed than responding T cells. Since the autologous MLR may be important in in vivo regulation of immune responses, its reduction by low-dose glucocorticoids may be of clinical relevance. This suppressive effect must be considered in studies of the autologous MLR in patients receiving glucocorticoid therapy.

It has been hypothesized that the selective recognition of tissue-specific endothelial cell molecules helps determine the in vivo distribution of lymphoid effector cells by controlling the extravasation of their circulating precursors. Here we report (a) immunofluorescence studies of the cell surface phenotype of human lamina propria lymphocytes (LPL), including staining with monoclonal antibody Hermes-1, which defines a 90-kDa lymphocyte surface glycoprotein involved in recognition of high endothelial venules (HEV); and (b) functional analyses of the ability of LPL to bind to HEV in frozen sections of mucosal lymphoid tissues (appendix or Peyer's patch) vs. peripheral lymph nodes. Essentially all LPL bear the Hermes-1 antigen, over 90% at levels comparable to those of circulating PBL. As a population, LPL display a quantitative preference for adherence to mucosal HEV, binding 0.8-1.5 times as well as PBL to mucosal HEV, but only 0.1-0.5 times as well to HEV in peripheral lymph nodes. Of particular interest was the behavior of the lymphoblast fraction, which typically constituted 3-7% of LPL. These cells, defined by size, consisted of a mixture of T cells and surface IgA+ blasts. One hundred percent were Hermes-1 bright, and they bound 4-8 times more efficiently to mucosal HEV than PBL while failing to bind detectably to lymph node HEV. LPL binding to mucosal HEV involves the gp90 Hermes, since the monoclonal anti-gp90 antibody, Hermes-3, and a polyclonal anti-gp90 antiserum inhibit the binding of small LPL and of LP blasts. The remarkable efficiency and specificity of binding by LP blasts may reflect retention of homing properties of the blood-borne precursors of these blasts and is discussed in relation to the capacity of immunoblasts in mesenteric nodes and in thoracic duct lymph to traffic selectively to mucosal lymphoid and extralymphoid sites. The demonstration of organ-specific endothelial cell recognition by LP lymphoblasts provides considerable support for the concept that selective interactions with endothelium play an important role in directing the distribution of activated lymphocyte subsets in vivo.

The mechanism of decreased lymphocyte responsiveness after major surgery is unclear. Because sodium pentobarbital, and intermediately long-acting barbiturate, will reproducibly induce anesthesia in experimental animals, we utilized a canine model to investigate its effect on lymphocyte proliferation induced by the mitogenic lectins erythroagglutinating phytohemagglutinin (E-PHA) and leukoagglutinating phytohemagglutinin (L-PHA). Although no effect was observed at 10 minutes or 1 hour after an anesthetic dose of sodium pentobarbital, after 1 and 3 hours of anesthesia, canine lymphocytes were significantly suppressed, as demonstrated by decreased responsiveness to E-PHA and L-PHA mitogen stimulation. After 3-hours the majority of animals had mitogenesis values of less than 50% of the preanesthetic control values. Recovery, as measured by a return to at least 70% of the preanesthetic mitogenesis value, was noted in the majority of animals at 24, 48, and 72 hours. In order to investigate the machanisms of the in vivo capability of sodium pentobarbital to induce immunosuppression of lymphocyte transformation, in vitro studies were carried out. Sodium pentobarbital was found to significantly inhibit mitogen-induced canine mononuclear cell blastogenesis at anesthetic (1.5 to 3.0 mg%) drug concentrations in vitro. Lymphocytes pretreated with barbiturate and washed prior to plating did not show this inhibiting effect. Our findings suggest that depression of the immune response reported in patients after operation could result from short-acting barbiturates administered during the induction phase of clinical anesthesia. Furthermore, the suppression may involve in vivo metabolism of pentobarbital, hormones or other in vivo factors, since washed lymphocytes from the in vivo but not the in vitro experiments demonstrated suppression. These results indicate that anesthesia may be an important factor in the immunosuppression reported after major surgery.

Characterization of cold reactive lymphocytotoxic antibodies present in sera from Thai adults with malaria revealed that the antibodies are predominantly 19S (IgM), directed against both autologous and allogeneic mononuclear cells, complement-dependent, present in titres ranging from 1:2 to 1:16, and exhibit greater lymphocytotoxic activity during the acute stage of malarial infection than during the convalescent stage. The lymphocytotoxic antibodies were primarily directed against B cell targets or both B as well as T cell targets. In addition some sera were reactive with enriched monocyte/macrophage indicator cells at 15 degrees C, but not 37 degrees C. Antibodies directed against B cell targets were lymphocytotoxic both at 15 degrees C as well as 37 degrees C. The results indicate that IgM lymphocytotoxic antibodies in the sera of patients with malaria are directed primarily against B cells with reactivity to a lesser extent against T cells and macrophages and thus may play an immunoregulatory function in the humoral immune response to malaria infection.

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 observed that canine peripheral blood mononuclear cells are ineffective as mediators of spontaneous cell-mediated cytotoxicity (SCMC) despite being excellent mediators of antibody-dependent cellular cytotoxicity (ADCC). Canine lymphocytes were unable to kill seven cell lines in SCMC assays including Chang, K-562, dog kidney, and foetal intestine. One the other hand, they were able to kill Chang and K-562 cell line cells as well as chicken red blood cells in ADCC assay systems. Canine mononuclear cells were 40% E-rosette forming, 30% surface immunoglobulin bearing, 14% Fc receptor bearing, and 13% esterase staining. K-562 cell line cells inhibited the capability of human peripheral blood cells but not canine peripheral blood cells to kill CRBCs in an ADCC assay. Fc-receptor bearing human lymphocytes of both T-cell and null-cell subclasses mediated both SCMC and ADCC, while Fc-receptor bearing canine lymphocytes mediated ADCC but not SCMC. These observations add to the evidence of a dichotomy between SCMC and ADDC with regards to either cell type or cell processes.

Calcium ionophore A23187 was found to induce human white blood cells to kill human red blood cells. Optimal conditions for ionophore-induced cellular cytotoxicity (IICC) included an 18 h time period, an incubation temperature of 25 degrees, a 25:1 or 50:1 killer:target cell ratio,and a final ionophore concentration of 2 . 5 microgram/ml. WBC or granulocytes which were either frozen and thawed three times or sonicated were capable of mediating IICC. As intact cells, granulocytes (67 . 2% cytotoxicity), monocytes (34 . 8%), B cells (22 . 0%) and Null cells (19 . 3%) were effector cells but T cells (7 . 4%) were not. After fragmenting these cells, all cell types including T cells were able to mediate IICC. When cell lines (K562, Chang, and NCTC) were used as effectors, none would mediate IICC when intact. After freezing and thawing, Chang and NCTC would not mediate IICC, whereas K562 cells did. These studies may be indicative of a calcium-dependent, membrane-localized mechanism in cellular cytotoxic processes, and may provide a useful indicator system for isolation of the enzyme systems involved in cellular cytotoxicity.

Elucidation of mechanisms involved in the control of colonic production of mucus requires direct examination of glycoprotein synthesis and secretion by colonic mucosa. In the past, the limited viability of intestinal mucosa in vitro has hampered such investigations. When maintained in an organ culture system, mucosal biopsies of rabbit colon and human rectum remained viable for 24 h as documented by morphologic appearance and a steady rate of protein synthesis and secretion. These biopsies also incorporated (14)C-labeled glucosamine into tissue glycoproteins and secreted labeled glycoproteins at a steady rate for 24 h. Glucosamine was predominantly incorporated into macromolecules that were ultimately secreted, in contrast to leucine, which was predominantly incorporated into tissue macromolecules. When studied by autoradiography, cultured rabbit colonic biopsies synthesized and secreted glycoproteins in vitro at cellular sites and over a time-course similar to those previously described for the intestine of intact animals. Acetylcholine consistently stimulated secretion of labeled glycoproteins but did not alter glycoprotein synthesis. In contrast, cycloheximide inhibited glycoprotein synthesis but had no effect on the secretion of newly synthesized glycoproteins. Rectal biopsies from patients with active ulcerative colitis incorporated increased amounts of [(14)C]glucosamine into glycoproteins during organ culture and secreted labeled glycoproteins more rapidly into the incubation medium when compared to biopsies obtained from healthy volunteers These findings indicate that organ culture provides a useful means of directly examining the synthesis and secretion of glycoproteins by healthy and diseased colonic mucosa.

The spontaneous synthesis and secretion of immunoglobulin by human bone-marrow mononuclear cells (MNC) in vitro, as well as its suppression by the addition of pokeweed mitogen (PWM), were previously reported by this laboratory. In the present study we demonstrate that this suppression is mediated by a soluble substance elaborated by marrow MNC stimulated with PWM. Marrow MNC were pulsed for 1 hr with PWM, washed and recultured for 7 days in media without PWM. The culture supernatants were collected by centrifugation and filter sterilized before addition to fresh marrow MNC in the in vitro antibody synthesis assay. The 14-day assay culture supernatants were then subjected to a solid phase radioimmunoassay to determine the immunoglobulin content. The suppressor substance was non-specific as to immunoglobulin isotype and was not genetically restricted. Suppressor activity was diminished by heating the supernatants at 56 degrees for 1 hr. The activity could be elaborated by cells subjected to 1000 R or 2000 R before or after 1-hr incubation with PWM. While the addition of PWM anytime during the culture period would suppress IgA production at the level produced up to that time, the suppressor substance only suppressed IgA production when added during the first 4 days of culture. The addition of indomethacin had no effect on the suppressor activity indicating that the activity was not mediated by prostaglandin. Including human fibroblast interferon or hydrocortisone in the assay cultures had no effect on IgA production or its suppression by PWM. We concluded that the lectin-induced suppression was mediated by a marrow-derived suppressor substance (MDSS).

A clone of natural killer (NK) cells (JTB18) was found to be ultrastructurally similar to peripheral blood large granular lymphocytes (LGL). These cells incorporated [35S]sulfate into cell-associated proteoglycan molecules, which were then isolated by CsCl density gradient centrifugation. As assessed by gel filtration chromatography, the native 35S-labeled proteoglycan and its beta-eliminated 35S-labeled glycosaminoglycans were of Mr approximately 200,000 and 50,000, respectively. The 35S-labeled proteoglycans were resistant to proteolysis, since their Mr were apparently not altered by incubation with either pronase or S. aureus V8 protease. The purified NK cell 35S-labeled proteoglycans were degraded by approximately 90% to 35S-labeled disaccharides with either chondroitinase ABC or AC. High performance liquid chromatographic analysis of the digests revealed these disaccharides to be composed entirely of chondroitin sulfate A (glucuronic acid----N-acetylgalactosamine-4SO4). Whole 35S-labeled cells incubated with chondroitinase ABC failed to release 35S-labeled disaccharides into the supernatant, and x-ray energy-dispersive analysis revealed that sulfur-containing molecules were present in the intracellular granules, thereby localizing the NK cell-associated proteoglycan primarily in the granules of the cell, rather than on the plasma membrane. The 35S-labeled cloned NK cells incubated for 30 min to 4 h with K562 tumor cell targets at a 0.5:1 ratio exocytosed a mean of 49% of the granular 35S-labeled proteoglycans during the first 60 min of the culture. Proteoglycan release was maximal with an effector/target cell ratio of 0.5:1 for JTB18:K562. Significant proteoglycan release from JTB18 NK cells was also obtained with other sensitive target cells such as REX, Molt4, and CEM, but not with cells such as KG1 and Laz156, which have been shown previously to be resistant to killing by this NK cell. Thus, protease-resistant intracellular proteoglycans with chondroitin sulfate A side chains are specifically exocytosed from the granules of human NK effector cells upon contact with sensitive targets, suggesting that these proteoglycans may be involved in the mechanism of cytotoxicity.

Peripheral T cell lymphoma developed in two patients several years after an initial clinical presentation of eosinophilia, pruritic skin rash, and vasculitis with lymphocytic infiltrates. Despite treatment with combination chemotherapy, the patients survived less than six months after the malignant lymphoma emerged. Immunologic characterization of tumor cells demonstrated features characteristic of peripheral T cell lymphoma. T lymphocytes from one patient had the uncommon phenotype of T3-negative, T4-positive, T8-negative. Extensive functional studies of this patient's lymphocytes revealed a poor proliferative response as well as an inability to help in immunoglobulin production, despite the preponderance of T4 lymphocytes. It is hypothesized that this syndrome is a consequence of the activity of products elaborated by neoplastic T cells.

To assess general cytotoxic effector cell capabilities by peripheral blood mononuclear cells from patients with active malaria infections, we examined antibody-dependent cellular cytotoxicity, spontaneous cell-mediated cytotoxicity, and lectin-induced cellular cytotoxicity by using human and chicken erythrocyte, Chang cell line, and K562 cell line targets. By using human erythrocyte and Change cell line targets, we found that Thai adults naturally infected with malaria had significantly impaired lectin-induced cellular cytotoxicity. In addition, spontaneous cell-mediated cytotoxicity was deficient with K562 but not with Chang cell line targets. Finally, no change in antibody-dependent cellular cytotoxicity was observed when chicken erythrocyte or Chang cell line targets were used. These observations, coupled with our previous observations of a physical loss of peripheral blood T cells, the presence of lymphocytotoxic serum antibodies, and defective T suppressor cell generation in patients with malaria, indicate that major alterations in the cellular immune system occur in patients with active malaria infections.

Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibit spontaneous cytotoxicity mediated by human natural killer (NK) cells. The aim of this study was to determine which stage(s) of the NK cytotoxic reaction is inhibited by these compounds. Effector/target cell binding studies performed in parallel with cytotoxicity assays using purified large granular lymphocytes indicated that inhibition is a post-binding event. The kinetic profile of inhibition in a calcium pulse assay showed that inhibition continues long after the effector cell triggering stage and that although sulfasalazine may have some inhibitory effect on the calcium-dependent events of the programming phase, sulfapyridine continues to inhibit during the calcium-independent or lethal hit phase of the cytotoxic sequence. The NK soluble cytotoxic factor (NKCF) assay was used as a measure of the lethal hit since the time course of this assay permits study of the various substages of this terminal event in the lytic sequence. Sulfasalazine and sulfapyridine but not 5-aminosalicylate inhibited NKCF-mediated target cell lysis. Different substages of the NKCF-induced lytic reaction were affected by these agents. Sulfasalazine appears to inhibit binding of NKCF to the target cell whereas sulfapyridine predominantly inhibits early post-binding events.

A large proportion of DNA synthesized in vitro by human lymphocytes stimulated with plant mitogens or specific antigens is selectively excreted from the cells. To determine if DNA excretion differs among various types of lymphocytes, we examined purified human lymphocyte subpopulations for DNA synthesis and excretion in response to stimulation by L-PHA. The relative proportion of newly synthesized DNA that is excreted by unseparated mononuclear cells, macrophage-depleted cells, T, and B lymphocytes is identical despite great differences in the magnitude of their responses. Low levels of both DNA synthesis and excretion by macrophage-depleted cells and B cells can be increased by reconstitution with macrophages and T cells, respectively. These data indicate that DNA exretion is a general property of lymphocytes stimulated to undergo DNA synthesis by plant mitogens.

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 Crohn's 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. Crohn's 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.

A black woman with chronic lymphocytic leukemia (CLL) was found to have monoclonal B lymphocytes with one type of surface immunoglobulin and one variant of G6PD (glucose-6-phosphate dehydrogenase) (G6PD A). Erythrocytes and T cells contained both G6PD A and G6PD B and hence were of polyclonal origin. The CLL cells in this patient likely arose from a developmental stage later than the step of differentiation into T and B lymphocytes. Furthermore, her erythrocytes did not arise from a stem cell affected by the CLL process.

Using Chang and K-562 cell line cells as targets, we have observed that a number of sugars are capable of inhibiting spontaneous cell-mediated cytotoxicity (SCMC) but not antibody-dependent cell-mediated cytotoxicity (ADCC). The sugars D(-)ribose, beta-gentiobiose, N-acetyl-D-galatosamine, and alpha-lactose all significantly inhibited SCMC of Chang and K-562 cell line cells. Because these same sugars caused no inhibition of ADCC against either Chang or K-562 cell line cells in assays run simultaneously, the results do not appear to be due to a non-specific toxic effect of the sugars against the effector cells. These studies add to the evidence that ADCC and SCMC are mediated by separate receptors. Furthermore, they provide evidence that endogenous lectin receptors or lectin-like molecules may be involved in the recognition and/or effector stages leading to SCMC. Thus, NK cells may recognize targets by virtue of receptors capable of interacting with monosaccharide, disaccharide, or oligosaccharide sequences present alone, as glycolipids, and/or as glycoproteins on the target cell surface.

Proteins binding the third component of complement (C3) were isolated by affinity chromatography from surface-labeled solubilized membranes of human peripheral blood cells and cell lines. The isolated molecules were subjected to NaDodSO4/PAGE, and autoradiographs of these gels indicated that C3-binding proteins could be divided into three groups based on Mr: (i) gp200, an approximately 200,000 Mr molecule previously identified as the C3b/C4b receptor or CR1; (ii) gp140, an approximately 140,000 Mr molecule previously identified as the C3d receptor or CR2; and (iii) gp45-70, a heretofore unrecognized group of 45,000-70,000 Mr C3-binding molecules. The cell distribution, Mr, antigenic cross-reactivity, and specificity of gp45-70 were examined. Erythrocytes have no detectable gp45-70, but all leukocyte populations examined possess this group of molecules. On neutrophils and mononuclear phagocytes, CR1 is the predominant C3-binding glycoprotein, but gp45-70 is present on both cell populations and on macrophage and neutrophil cell lines. B plus null cells, chronic lymphocytic leukemia cells, and an Epstein-Barr virus-transformed B-cell line possess CR1, CR2, and gp45-70. On T cells and T-cell lines gp45-70 is the predominant or, in some cases, the only C3-binding protein isolated. gp45-70 is structurally characterized as a broad band or doublet with a mean Mr that is slightly different for each cell population. gp45-70 binds iC3, C3b, and C4b, but not C3d, indicating that the binding region is probably within the C3c portion of C3b. A polyclonal antibody to CR1 and monoclonal antibodies to CR1 and CR2 do not immunoprecipitate gp45-70. While gp45-70 has not been previously characterized on human cells, a C3b-binding glycoprotein of similar Mr is present on rabbit alveolar macrophages. We conclude that gp45-70 is an additional group of membrane proteins present on human leukocytes that possess ligand-binding activity for C3b.