Dapsone suppresses integrin-mediated neutrophil adherence function.

The anti-inflammatory influence of dapsone may involve suppression of neutrophil chemotaxis to selected attractants, but other actions of the drug are likely also involved. We have discovered that dapsone may suppress migration of neutrophils to extravascular sites through inhibition of adherence functions required for neutrophil recruitment. Neutrophil adherence mediated by integrins (CD11/CD18 or Mac-1 family receptors) was measured in vitro in terms of binding of stimulated cells to albumin-coated wells of microtiter plates, using phorbol myristate acetate (PMA) and N-formylmethionyl-leucyl-phenylalanine (FMLP) as stimuli. Adherence was assessed by staining attached cells with crystal violet dye and measuring the dye concentration at OD590 using an automated plate reader. The role of integrins in this assay was confirmed by the ability of anti-integrin antibody to suppress stimulated neutrophil adherence. The OD590 value for cells adhering to albumin in the absence of stimulus and dapsone averaged 0.2 +/- 0.04 (SEM) over five experiments. In the presence of 0.1 microM PMA or 10(-6) M FMLP, the OD590 values averaged 0.88 +/- 0.1 and 0.75 +/- 0.12, respectively. Dapsone did not affect unstimulated neutrophil adherence but, when present with stimulus, produced a dose-related inhibitory effect on adherence. Fifty percent inhibitory doses were approximately 150 micrograms/ml dapsone for both stimuli. Sulfapyridine reproduced the inhibitory effect of dapsone, but two structurally related compounds, hydrochlorothiazide and furosamide, did not. The observed ability of dapsone to inhibit neutrophil chemotaxis under agarose to FMLP and interleukin-8 may also be explained by interference with integrin-mediated adherence required for motility in this assay system. To consider if dapsone might have a similar inhibitory influence on neutrophil adherence in vivo, we tested the stimulated adherence function of neutrophils isolated from three individuals on dapsone therapy for dermatitis herpetiformis. Stimulated adherence of patients' cells averaged less than 40 percent of the control value. Suppression of leukocyte integrin function may therefore also contribute to the ability of dapsone to inhibit neutrophil infiltration in neutrophilic dermatoses.

Sialomucin and lytic susceptibility of rat mammary tumor ascites cells.

The potential role of cell surface sialomucin in preventing natural killer (NK)-mediated lysis of tumor cell targets has been addressed by comparing the properties of 2 NK-resistant [ascites (ASC) and short-term cultured (STC)] and 2 NK-susceptible [tunicamycin-treated (TUN) and long-term cultured (LTC)] preparations of 13762 MAT-B1 rat mammary tumor cells. Both the ASC and STC cell preparations contain elevated levels of the sialomucin ASGP-1 relative to TUN and LTC preparations as determined by [3H]glucosamine labeling and by binding of peanut agglutinin. The major difference in the susceptibility to NK-mediated lysis appeared to be due to the differences in the susceptibility to lysis by lytic granules, rather than to differences in the ability to bind or trigger effector cells, since TUN and LTC cells were approximately 10-fold more sensitive to lysis by lytic granules than were ASC and STC cells. All preparations inhibited the lysis of the susceptible target YAC-1 by normal rat splenocytes, indicating an ability to bind these effector cells. Triggering of effectors, as monitored either by incorporation of 32P into phosphatidylinositol or by transmethylation of phosphatidylcholine, was similar for the positive control YAC-1, STC, TUN, and LTC, whereas ASC appeared to be defective in triggering effectors. These results suggest that tumor sialomucin blocks the final phase of lysis, but not the initial recognition of tumor cells by NK effectors.

Effect of tunicamycin on sialomucin and natural killer susceptibility of rat mammary tumor ascites cells.

The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma contain a dominant cell surface "complex" consisting of two glycoproteins: ascites sialoglycoprotein (ASGP)-1, a Mr 600,000-700,000 peanut agglutinin-binding sialomucin, and ASGP-2, a Mr 120,000 concancavalin A-binding glycoprotein (Sherblom et al., J. Biol. Chem., 255: 783-790, 1980; Sherblom and Carraway, J. Biol. Chem., 255: 12051-12059, 1980). Although both cell lines are resistant to lysis by natural killer cells, treatments which result in loss of cell surface ASGP-1 render the cells susceptible to natural killer cell lysis (Sherblom and Moody, Cancer Res., 46:4543-4546, 1986). Treatment of the ascites cells with 5 micrograms/ml tunicamycin for 24 h effectively inhibits glycosylation of ASGP-2 without affecting cell viability or total protein synthesis. Under these conditions, expression of ASGP-1 is depressed by at least 50% in both cell lines, as monitored by [3H]glucosamine incorporation and by binding of peanut agglutinin to intact cells. The size distribution of O-linked oligosaccharides in ASGP-1 from tunicamycin-treated versus control MAT-B1 cells is indistinguishable, as determined by Bio-Gel P-4 chromatography following alkaline-borohydride treatment. Complex isolated from either treated or control cells bands at the same density in a CsCl gradient containing Triton X-100 and contains a diffuse band corresponding to ASGP-2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tunicamycin-treated cells, consistent with the reduced expression of ASGP-1, are significantly more susceptible to natural killer cell-mediated lysis, when compared to untreated controls. The results suggest that N-linked glycosylation is a prerequisite for sialomucin synthesis and/or complex formation.

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein).

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479-81; Hession et al., (1987) Science 237:1479-84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547-53]. We report here that the Man6(7)GlcNAc2-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2-2 microM, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man9GlcNAc2-R the most inhibitory of those tested [Muchmore et al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man5GlcNAc2-R and Man6GlcNAc2-R. Man7GlcNAc2-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).

Bovine serum sialic acid: age-related changes in type and content.

1. The sialic acid content of newborn calf serum (4.8 mumol/ml) is approx. 3-fold higher than that of mature animals (1.4 mumol/ml) and decreases to 2.4 mumol/ml at 20 days of age. Colostrum-fed and colostrum-deprived calves have similar levels of sialic acid from birth to 14 days of age. 2. The high level of sialic acid in newborn calf serum is due predominantly to N-acetylneuraminic acid, since this sialic acid accounts for 93% of the total and since less than 5% of the sialic acid is O-acetylated. 3. Comparison of day 0 and day 20 serum by gel filtration and by SDS polyacrylamide gel electrophoresis demonstrates that the increase in sialic acid is associated with increased production and/or sialylation of components with MW of 45-60 kDa. 4. A high percentage (64%) of the sialic acid in newborn calf serum is detected with the lipid-linked sialic acid assay, relative to 20 day old (25%) or mature (18%) animals. 5. This indicates that the glycoproteins of newborn calf serum are more efficiently extracted under the conditions of this assay than glycoproteins of mature serum.

Immunosuppression, sialic acid, and sialyltransferase of neonatal and maternal bovine serum.

Serum samples of 7 cows from -10 to +10 days following parturition and of 7 calves from 0 to 20 days following birth were tested for the ability to inhibit mitogen-stimulated proliferation of lymphocytes, for cortisol and progesterone concentrations, and for sialic acid and sialyltransferase activity. Calf serum inhibited phytohemagglutinin (PHA)-stimulated proliferation of lymphocytes, with maximal inhibition at 12-24 h following birth, whereas no consistent immunosuppressive activity was detected in the maternal serum. Sialic acid was greatly elevated in calf serum (4.8 +/- 0.2 mumol/ml) relative to adult control values (1.3 +/- 0.1) and decreased continuously from day 0 to day 20. Sialic acid of maternal serum was slightly elevated prior to parturition (1.7 +/- 0.1) and increased to peak at 2.5 +/- 0.1 on day 8 following parturition. Sialyltransferase of both maternal and calf serum increased dramatically following parturition to peak at day 2. For calf serum, a moderate correlation was observed between sialic acid and cortisol concentration (r = 0.71) and between sialic acid and suppression of PHA-stimulated proliferation (r = 0.60). The results demonstrate that serum of 12-24 h-old calves is immunosuppressive in vitro, and suggest that changes in sialic metabolism may accompany cortisol-related immunosuppression in these animals.

Cell surface sialomucin and resistance to natural cell-mediated cytotoxicity of rat mammary tumor ascites cells.

MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma both contain sialomucin as a major cell surface component and are resistant to cytolysis by normal rat spleen lymphocytes [3 +/- 2% (SD) and 0 +/- 1%, respectively]. Susceptibility to lysis did not increase following treatment of cells with neuraminidase, fucosidase, or alpha- or beta-galactosidase. Treatment with trypsin significantly increased the susceptibility of MAT-B1 (14 +/- 3%) but not MAT-C1 (5 +/- 2%). Following 1 month in culture, the sialomucin content of MAT-B1 cells dropped from 30% to 8% (determined by glucosamine labeling) and natural cell-mediated cytolysis increased to 16 +/- 4%, whereas the sialomucin content and susceptibility of MAT-C1 cells did not change. The results indicate that the relatively minor changes associated with removal of cell surface sialic acid or fucose residues do not result in increased susceptibility of the ascites cells to cytolysis. However, susceptibility of MAT-B1 cells to lysis by normal rat spleen lymphocytes was inversely correlated with the amount of major glycoprotein (r = -0.96).

Sialyltransferase of bovine serum: age- and hormone-related changes.

Sialyltransferase activity of bovine serum with the acceptor asialofetuin exhibits a pH optimum at 6.0-6.5, no divalent cation dependence, and a Km for CMP-sialic acid of 0.05 mM. Although a 2-fold increase in sialyltransferase activity with the acceptor asialofetuin is observed in serum samples from 2-day-old vs 20-day-old calves, the relative activity towards other glycoprotein acceptors is not different between the groups. With the acceptor lactose, the major product (greater than 95%) for all samples is 3'-sialyllactose, suggesting that the elevated levels of sialyltransferase in 2-day-old calves are due to Gal-R (alpha 2-3) sialyltransferase.

Immunosuppression, sialic acid, and sialyltransferase of bovine serum as a function of progesterone concentration.

Serum samples from progesterone-oestrogen-treated ovariectomized Holstein cows (N = 4) were compared with samples from control ovariectomized Holstein cows (N = 4) to determine the effects of physiological levels (0-6 ng/ml) of circulating progesterone. The average progesterone level in treated animals rose from 1 ng/ml (Day 0) to plateau at 5 ng/ml (Days 12 to 36). Sera from progesterone-oestrogen-treated cows during Days 4 to 10 significantly suppressed stimulation of lymphocytes by phytohaemagglutinin as compared with sera from control cows (P = 0.02), whereas no differences were detected in serum samples from Days 12 to 36. Serum samples from progesterone-oestrogen-treated or control cows did not affect the stimulation of lymphocytes by pokeweed mitogen. Sialyltransferase activity (P = 0.0002) and sialic acid content (P = 0.006) were both significantly elevated in serum from progesterone-oestrogen-treated animals compared with controls during Days 8 to 16, whereas no significant differences were observed at later times. The results suggest that suppression of phytohaemagglutinin-induced stimulation, sialic acid content, and sialyltransferase activity are sensitive not to the circulating level of progesterone but rather to increases in progesterone concentration, with maximal effects observed at Days 8, 12 and 12, respectively, after the start of progesterone treatment. The work provides a preliminary basis for further studies on the mechanism of immunosuppression by steroids and during pregnancy.

Non-specific cytotoxic activity of teleost leukocytes.

The existence of lymphoid cells with "natural" killer activity in mammals and birds has been known for some time. Several previous reports have demonstrated similar activity in carp and catfish kidney leukocytes. However, the activity previously reported was directed towards established mammalian cell lines. In this report we confirm the existence of spontaneous killer activity in other species of teleosts, including salmonids. This spontaneous cytotoxic activity is directed towards several established teleost, as well as mammalian, cell lines. Cytotoxic activity appears to be optimal at 20 degrees C in an 8 hour 51Cr release assay. The RTG-2, AS, GS and EPC cell lines of teleost origin are susceptible to lysis by kidney, spleen, and blood leukocytes of Salmo salar, Salmo gairdneri, and Notemigonus crysoleucas. Furthermore, the susceptibility of the RTG-2 and AS teleost cell lines to killing by kidney leukocytes of both S. salar and S. gairdneri was significantly enhanced by preinfection of the target cells with infectious pancreatic necrosis virus.

Studies on the syngeneic mixed lymphocyte reaction. III. Development of a monoclonal antibody with specificity for autoreactive T cells.

Monoclonal antibodies with specificity for autoreactive murine T cells have been developed. These antibodies inhibit proliferative response of splenic T cells activated by syngeneic spleen cells. These antibodies have no effect on the proliferative response of T cells activated by allogeneic spleen cells or PHA. The number of splenic T cells that react with these monoclonal antibodies is comparable in several normal mouse strains.

The developmental biology of the syngeneic mixed lymphocyte reaction.

The syngeneic mixed lymphocyte reaction (SMLR) is the proliferative response of T lymphocytes cultured with non-T lymphocytes. Normal mouse serum supports proliferative activity in the SMLR comparable to levels observed when fetal calf serum is used. Thus, the SMLR does not appear to be a response to xenogeneic antigens. The ontogeny and senescence of the SMLR was also studied. The SMLR is impaired in mice less than 4 weeks of age but attains adult levels of activity at 4 weeks of age. The SMLR is also impaired in 24-month-old mice. These impairments were observed regardless of the source of serum used in the cultures. Finally, monoclonal antibodies have been developed which identify a subpopulation of spleen cells which respond in the SMLR. In indirect immunofluorescence these antibodies stain 50-80% of cells activated in the SMLR, but only 5-8% of spleen cells or cells activated in the allogeneic MLR. These antibodies block the proliferative response in the SMLR but do not interfere with the response to alloantigens or PHA.

Lymphocyte transformation induced by autologous cells. XV. Xenoantigens are not required for the proliferative response observed in the autologous mixed lymphocyte reaction.

The autologous mixed lymphocyte reaction is the proliferative response of T cells cultured with autologous non-T cells. This reaction has been described as being immunological in nature, i.e., possessing memory and specificity. The generation of T cells with regulatory and effector function has also been reported during the course of this reaction. More recently it has been reported that the proliferative response observed is due to the exposure of T cells to xenoantigens used in separating T and non-T cells. We have found that the presence of antigens such as sheep erythrocytes and fetal calf serum is not required for the induction of proliferation in T cells by autologous non-T cells, although exposure to such antigens may augment [3H]thymidine incorporation. In certain individuals who are sensitized to these xenogeneic antigens, the proliferative response of their lymphocytes in the autologous mixed lymphocyte reaction is very greatly enhanced if exposed to xenoantigens.

Lymphocyte transformation induced by autologous cells. XII. Quantitative and qualitative differences between human autologous and allogeneic reactive T lymphocytes.

The T cell populations that respond to autologous or allogeneic non-T cells are distinct, at least to a considerable extent. This conclusion is based on the differences in a) the size of the two populations; on the average one in 260 (individual frequency range, 1:149 to 1:417) T cells respond to allogeneic non-T cells whereas one in 3100 (individual frequency range, 1:2200 to 1:5000) T cells respond to autologous non-T cells; b) the phenotype of T cells activated in the MLR; autoactivated T cells were drawn predominantly from the OKT4-reactive T cell subpopulation, whereas alloactivated T cells activated in the allogeneic MLR were drawn from the OKT4, OKT5, and OKT8-reactive T cell subpopulations; and c) the specificity of activated and nonactivated T cells obtained on Percoll gradients after autologous or allogeneic MLR. Auto- or allodepleted T cells recovered from the 55 to 60% Percoll interface retained full alloreactivity or autoreactivity, respectively. However, auto- or alloactivated T cells recovered from the 40 to 50% Percoll interface showed secondary kinetics to allogeneic and autologous non-T cells. Whether the alloreactivity or autoreactivity found in autoactivated or alloactivated T cells, respectively, represents true cross-reacting T cells or T cells nonspecifically recruited during the MLR cannot at present be decided.

Lymphocyte transformation induced by autologous cells. XI. The effect of age on the autologous mixed lymphocyte reaction.

The autologous mixed lymphocyte reaction (MLR) was lower in newborn infants and healthy subjects over 65 years of age than in adults between the ages of 20 and 32. In contrast, the allogeneic MLR, although impaired in newborn infants, was normal in elderly subjects. The degree of impairment of the autologous MLR in elderly subjects was correlated with the impairment in the response of lymphocytes from elderly subjects to phytohaemagglutinin (PHA) and Staphylococcus aureus proteins A (SPA). The percentage of autorosetting T cells and of T cells with the OKT4 phenotype was increased in elderly subjects. These findings are paradoxical as autoreactive T cells in young adults have been reported to be drawn from these two T-cell subpopulations.

Lymphocyte transformation induced by autologous cells. X. Soluble factors that generate cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) were not generated in an autologous mixed lymphocyte reaction (MLR) in the presence of human AB serum. No CTL were generated in cultures that contained T cells and irradiated autologous, allogenetic, or hapten-modified autologous T lymphocytes. However, when allogeneic T lymphocytes or hapten-modified autologous T lymphocytes were added to an autologous MLR, specific CTL were generated. Furthermore, supernatant medium from an autologous MLR allowed the generation of specific CTL in T cell cultures to which allogeneic T cells or hapten-modified autologous T cells were added.

Lymphocyte transformation induced by autologous cells. VIII. Impaired autologous mixed lymphocyte reactivity in patients with acute infectious mononucleosis.

The autologous mixed lymphocyte reaction (MLR) is severely impaired in patients with acute infectious mononucleosis. Reactivity returned during the course of convalescence. The allogeneic MLR was not impaired in these patients. B cells from patients with infectious mononucleosis do not stimulate autologous T-cell proliferation, and this observation appears to explain the cellular basis of the impaired autologous MLR in infection. Two explanations for the B-cell defect were considered: (a) the influence of serum factors on B-cell function and (b) the effect of Epstein-Barr virus infection.