Involvement of interleukin 2 receptors in conceptus-derived suppression of T and B cell proliferation in horses

The mechanism by which a horse conceptus-derived immunosuppressive factor (HCS) of M(r) > 100,000 inhibits lymphocyte proliferation was investigated. The factor was obtained from the culture supernatants of 20-day-old horse conceptuses; activity, identified by reduced uptake of [3H]thymidine by mitogen-stimulated lymphocytes, was greatest (P < 0.01) in cultures stimulated by mitogen from pokeweed. HCS also suppressed cell proliferation stimulated by phytohaemagglutinin (P < 0.01), but had no effect on lipopolysaccharide-stimulated cells (P > 0.05). Data from a fluorescence-activated cell sorter indicated that supplementation with HCS reduced the number of T cells in phytohaemagglutinin-stimulated cultures and suppressed proliferation of T and B cells in pokeweed-mitogen-stimulated cultures compared with controls. Cell proliferation was greater (P < 0.01) in cultures supplemented with HCS 24 h after stimulation than in those treated at the start of stimulation, and was even greater (P < 0.01) when cells were treated 48 h after stimulation. The removal of HCS from treated lymphocyte cultures resulted in complete recovery of cell responsiveness, and stimulated proliferation of treated cells did not differ (P > 0.05) from that of control cells. The addition of stimulated equine lymphocyte supernatant to cultures supplemented with HCS did not significantly increase (P > 0.05) cell proliferation in response to pokeweed mitogen. Addition of recombinant human interleukin 2 (rIL-2) to HCS-treated cultures did not alter the suppressive activity of HCS, although cell proliferation was greater in cultures supplemented with rIL-2 than in controls (P < 0.01). HCS inhibition of IL-2 receptor (IL-2R) function was investigated using an IL-2-dependent murine cytolytic T lymphocyte cell line; the fraction of HCS of M(r) > 100,000 had no effect (P > 0.05) on proliferation of IL-2-dependent murine cytolytic T lymphocyte cells induced by rIL-2. Together, these data suggest that HCS suppresses proliferation of T lymphocytes during the early stages of cell activation by inhibiting IL-2R interaction and that this suppression interferes with interactions between T cells and B cells, thereby also indirectly inhibiting proliferation of B cells. The potent immunosuppressive capacity of HCS may be one factor responsible for inhibiting cell-mediated fetal allograft rejection during pregnancy.     

Rat natural killer cell, T cell and macrophage functions after intracerebroventricular injection of SNC 80

We investigated the effects of (+)-4-[(alpha R)-alpha-((2S, 5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N, N-diethylbenzamide (SNC 80), a nonpeptidic delta-opioid receptor-selective agonist, on rat leukocyte functions. Intracerebroventricular injection of SNC 80 (20 nmol) in Fischer 344N male rats did not affect splenic natural killer cell activity compared with intracerebroventricular saline-injected controls. SNC 80 also had no effect on concanavalin A-, anti-T cell receptor-, interleukin-2- and anti-T cell receptor + interleukin-2-induced splenic and thymic lymphocyte proliferation in most experiments. In some experiments, however, SNC 80 significantly (P < .01) caused a 41 to 93% increase of concanavalin A-, anti-T cell receptor-, interleukin-2- and anti-T cell receptor + interleukin-2-induced splenic lymphocyte proliferation compared to controls. Additionally, SNC 80 did not significantly affect splenic T cell or natural killer cell populations as measured by the expression of T cell receptoralphabeta, and T helper (CD4), T suppressor/cytotoxic (CD8) and natural killer cell surface markers. Finally, SNC 80 did not affect interferon-gamma- or lipopolysaccharide (LPS)-induced splenic nitric oxide, and LPS-induced tumor necrosis factor-alpha production by splenic macrophages. These results suggest that SNC 80 could be useful in the treatment of pain without suppressing immune function. However, the potential immunoenhancing properties of SNC 80 may be also valuable in immunocompromised individuals.     

Interactive effects of cocaine and gender on thymocytes: a study of in vivo repeated cocaine exposure

This study used a mouse model including both sexes to assess the impact of repeat cocaine exposure on the differentiation and function of T cell in thymus. Cocaine hydrochloride in 0.9% saline, 5 mg or 40 mg/kg, was administrated by i.p. injection to C57BL/6 mice for 10 days. Thymocytes were obtained 24 h after the 10th injection. Repeat in vivo cocaine exposure inhibited the proliferation of T lymphocytes in response to Con-A and Con-A plus anti-CD28. The proliferation induced by IL-2 in the Con-A stimulated T blasts was attenuated in cocaine treated mice. These effects were seen at a lower cocaine dose in female mice. The total number of thymocytes was reduced. Although the percentage of mature thymocytes (CD4+ CD8- and CD4- CD8+ cells) was not altered, the absolute cell numbers were attenuated. Both percentage and absolute cell number of immature thymocytes (CD4+ CD8+) decreased and the pre-mature (CD4- CD8-) cells increased. CD28 and CD25 expression were attenuated in Con-A stimulated thymocytes of mice treated with cocaine at 40 mg/kg. Interleukin 2 production was not significantly altered, however, gamma-IFN production was decreased by cocaine exposure at 40 mg/kg. In conclusion, cocaine exerts inhibitory effects on the function of mature thymocytes, and on the differentiation of thymocytes. A gender difference in response to cocaine was noted in that female mice were more sensitive to lower dose of cocaine exposure.     

Tumor growth alters T cell and macrophage production of and responsiveness to granulocyte-macrophage colony-stimulating factor: partial dysregulation through interleukin-10

Tumor growth induces phenotypic and functional changes among splenic T cells and macrophages (M phi) that contribute to the immunosuppression observed in tumor-bearing hosts (TBH). These changes partly arise through alterations in immune cell production of and responsiveness to cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important T cell- and M phi-derived cytokine that is produced during normal host immunogenic challenge, but it's involvement during cancer is poorly defined. In contrast, interleukin-10 (IL-10) is an inhibitory cytokine that is produced by immune cells as a deactivation factor. IL-10 can disrupt GM-CSF synthesis and may be associated with tumor-induced changes in cytokine synthesis. We determined if tumor growth alters T-cell and M phi synthesis of and responsiveness to GM-CSF, and if these alterations occur because tumor growth heightens immune cell sensitivity to IL-10. Tumor growth significantly decreased T-cell synthesis of GM-CSF during activation by concanavalin A, and TBH T cells were more susceptible to GM-CSF synthesis inhibition by IL-10 than their normal host (NH) counterparts. This suppression was observed using both unseparated splenic lymphocyte preparations and purified CD4+ and CD8+ T cells. Similarly, TBH M phi (both splenic and peritoneal) produced less GM-CSF than NH M phi during activation by lipopolysaccharide. Tumor growth also altered major histocompatibility complex (MHC) class II- M phi GM-CSF synthesis. TBH M phi were more susceptible to GM-CSF synthesis inhibition by IL-10 than their NH counterparts. Although TBH T cells demonstrate less proliferation than NH T cells during activation, tumor growth did not compromise T-cell responsiveness to GM-CSF. However, tumor growth did increase TBH T-cell susceptibility to inhibition of proliferation by IL-10. Tumor growth suppressed M phi responsiveness to GM-CSF, and IL-10 further decreased M phi responsiveness to GM-CSF. Collectively, these results suggest that T cell and M phi production of and responsiveness to GM-CSF is disrupted during tumor growth, and that TBH T cells and M phi are more susceptible to the suppressor activity of IL-10 than their NH counterparts.     

Inhibition of vascular smooth muscle cell growth by inhibition of fibronectin matrix assembly

The regulation of vascular smooth muscle cell (VSMC) proliferation by the fibronectin matrix was tested by treating human umbilical artery smooth muscle cells (HUASMCs) with a recombinant fragment of fibronectin (protein III1-C) that has previously been shown to modulate fibronectin matrix assembly. III1-C inhibited HUASMC proliferation by 75% to 90%. The inhibition of growth was time dependent; III1-C had no effect on DNA synthesis after 0 to 5 hours of treatment but did have an effect at 24 hours and beyond. III1-C did not stimulate apoptosis in these cells, indicating that the inhibition of proliferation was not due to an induction of programmed cell death. The effects of III1-C on cell growth were only specific for normal diploid smooth muscle cells. III1-C had no effect on the proliferation of IMR-90 fibroblasts, endothelial cells, NIH 3T3 cells, or the rat aortic smooth muscle cell line A7r5. However, III1-C did inhibit proliferation by primary rat aortic smooth muscle cells. An analysis of HUASMC fibronectin receptor (integrin alpha5beta1) distribution revealed that III1-C did not inhibit alpha5beta1 localization to focal contacts. Moreover, III1-C had no effect on the relative expression levels of seven different integrin subunits on HUASMCs. However, III1-C did inhibit fibronectin matrix assembly by rat aortic smooth muscle cells, HUASMCs, A7r5 cells, IMR-90 cells, and endothelial cells. An analysis of fibronectin synthesis indicated that the inhibition of fibronectin matrix assembly by III1-C was not due solely to a decrease in fibronectin synthesis. Finally, treatment of HUASMCs with anti-fibronectin monoclonal antibody L8 (which is known to inhibit fibronectin matrix assembly) also decreased the rate of HUASMC DNA synthesis. These results demonstrate that III1-C inhibits VSMC proliferation and suggest that this effect may be mediated by the inhibition of fibronectin matrix assembly.     

Cross-reactive memory T cells for Epstein-Barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex-bound peptide by T cells and its role in alloreactivity

In the present report, cytotoxic T lymphocyte (CTL) clones are described that display dual specificity for one of two common human leukocyte antigens (HLA B14 or B35) as alloantigens, and an immunodominant epitope (FLRGRAYGL) from Epstein-Barr virus (EBV) that binds to HLA B8. These T cell clonotypes were isolated from several unrelated HLA B8+, EBV-exposed individuals, and each distinct cross-reactivity pattern was associated with a common, public T cell receptor (TCR). In some individuals, CTL cross-reactive with these alloantigens completely dominated the memory response to this EBV epitope. Moreover, these memory T cells to EBV could be reactivated as a significant component of the repertoire of CTL responding to allogeneic stimulator cells expressing either HLA B14 or B35. These data illustrate how a history of infection with an immunogenic virus such as EBV can augment responsiveness to particular alloantigens; such influences may underlie the observed clinical association between herpesvirus infection and both allograft rejection and graft-versus-host disease. We have also explored the molecular basis for T cell cross-reactivity with alloantigens using the HLA B35 allo-reactive CTL clonotype. To elucidate the structural features of peptides that may be cross-recognized by these T cells, mono-substituted analogs of the viral epitope were screened for recognition, revealing broad specificity for major histocompatibility complex (MHC)-bound peptide. Based on the particular amino acid changes tolerated by the CTL at each peptide position, the human protein sequence database was searched for possible sequences that were recognized in association with HLA B35. Four peptides were identified (MPEATVYGL, IPIAPVYGM, KPSPPYFGL, and KPIVVLHGY) that were powerful activating ligands for the CTL when presented on HLA B35 but not B8. Thus, equivalent epitopes, capable of fully activating a single TCR, were formed by peptides with minimal obvious sequence homology bound to either HLA B8 or B35. These data indicate that degenerate peptide recognition by TCR may play an important role in the vigorous response of self-MHC-restricted T cells to alloantigens.     

Effects of lindane exposure on rainbow trout (Oncorhynchus mykiss) immunity. III. Effect on nonspecific immunity and B lymphocyte functions

The effect of the organochlorine insecticide lindane (gamma-hexachlorocyclohexane) was examined on some major immune functions of rainbow trout (Oncorhynchus mykiss) on the chemiluminescent response of pronephric cells (PMA-induced) in phagocytosis, on the proliferation of lymphocytes with B and T mitogens, and on the number of B lymphocytes analyzed by cytofluorometry. Two different methods of exposure were tried via food (first protocol) and via a single intraperitoneal injection (second protocol). After the oral contamination at a daily body dose of 1 mg/kg for 30 days, a decreased chemiluminescent response was observed with persisted for two more weeks and disappeared over 1.5 months. No effect was observed on lymphocyte proliferation and on the number of circulating B lymphocytes. In the second protocol lindane was administered intraperitoneally at 10, 50, or 100 mg/kg body wt. After 45 days the lymphocyte proliferation of B cells was depressed but not the T cell one. The B cells number in head kidney as measured by cytofluorometry was not significantly modified. Some nonspecific immunity parameters in sera were significantly modified.     

Signaling via major histocompatibility complex class II molecules and antigen receptors enhances the B cell response to gp39/CD40 ligand

Activated T cells induce proliferation and differentiation of resting B cells in vitro through their CD40 molecules and lymphokine receptors. However, despite constitutive B cell expression of CD40 and lymphokine receptors, widespread nonspecific polyclonal B cell activation by activated T cells is seldom observed in vivo. The present study was designed to test the hypothesis that signals delivered via the B cell antigen (Ag) receptor (membrane immunoglobulin, mIg) and major histocompatibility complex (MHC) class II molecules enhance B cell responsiveness to CD40-mediated signals, providing specificity to the Ag-nonspecific, MHC-unrestricted CD40 signal. To test this hypothesis, both an Ag-specific mouse B cell clone CH12.LX, and freshly isolated resting splenic B cells were cultured with either soluble or membrane-bound forms of the T cell ligand for CD40 (CD40L), in the presence or absence of additional signals provided by Ag or anti-IgM, interleukin-4, and class II-specific monoclonal antibody (mAb). Differentiation of CH12.LX cells and proliferation of splenic B cells in response to both forms of CD40L was greatly enhanced by exposure to mIg-mediated signals, with greatest enhancement seen when cells were cultured with Ag prior to receiving other signals. Response to CD40L was further enhanced by concurrent culture with class II-specific, but not class I-specific mAb. Enhancement was greatest at limiting concentrations of CD40L. The ability of class II MHC-mediated signals to enhance Ag-specific B cell responsiveness to CD40-mediated signaling may selectively promote the activation of B cell clones capable of cognate interactions with helper T cells.     

Lead stimulates lymphocyte proliferation through enhanced T cell-B cell interaction

We have studied the in vitro effects of lead (Pb) as Pb-acetate (0. 1-1000 ppm) on the activation of rat spleen (SP) cells. At a concentration of 0.5 to 200 ppm, Pb augmented the uptake of [3H]thymidine, progression of SP cells through the cell cycle, and allogeneic and syngeneic mixed lymphocyte reactions. However, at concentrations above 200 ppm, Pb inhibited the proliferation of these cells. To understand the cellular and molecular basis of these responses, we examined the effects of Pb on the proliferation of isolated T and/or B cell populations. Pb failed to stimulate the proliferation of isolated T and B cells; however, the addition of gamma-irradiated B cells to T cell cultures or irradiated T cells to B cell cultures resulted in Pb-induced incorporation of [3H]thymidine. On the other hand, macrophages were unable to reconstitute this response. Pb also induced a significant rise in the intracellular concentration of inositol 1,4,5-trisphosphate in SP cells; however, unlike the activation of lymphocytes through the antigen receptors, Pb did not significantly stimulate protein tyrosine kinase activity. These observations suggest that Pb facilitates the T cell-B cell interaction-dependent proliferation of lymphocytes through a signaling pathway(s) independent of the antigen receptor.     

IL-12 prevents neonatal induction of transplantation tolerance in mice

We investigated the effect of IL-12 on the induction of transplantation tolerance by neonatal injection of allogenic cells. We first observed that injection of newborn BALB/c mice with IL-12 and (A/J x BALB/c)F1 spleen cells prevented the Th2 alloimmune response induced by neonatal inoculation of F1 cells alone and allowed the differentiation of T cells secreting high amounts of IL-2 and IFN-gamma in mixed lymphocyte cultures with donor-type stimulators. Furthermore, IL-12 administration resulted in the emergence of anti-donor cytotoxic T lymphocyte responses although at lower levels than in control uninjected mice. In parallel, we found that mice injected at birth with IL-12 and F1 cells did not develop chimerism and were able to reject a donor-type skin graft as efficiently as control mice. We conclude that IL-12 inhibits the Th2 polarization of the newborn response to alloantigens and prevents thereby the establishment of transplantation tolerance.     

Stress-induced alteration of T-lymphocyte subsets and humoral immunity in mice.

Effects of acute rotational stress on the tissue distribution of lymphocyte subpopulations, and the capacity to mount a humoral immune response were examined in normal and adrenalectomized mice. The splenic lymphocyte population showed a significant alteration in its content of T lymphocytes in normal, but not adrenalectomized animals subjected to rotational stress. Thus adrenal steroids may influence lymphocyte subpopulations in animals responding to stress. Humoral immune responsiveness of splenic lymphocytes was assessed by a direct lymphocyte plaque forming cell assay following immunization with sheep erythrocytes. The peak number of lymphocytes forming antibody to sheep erythrocytes found in spleens of animals subjected to stress begun 24 hours after antigen inoculation was 50% of the number observed in animals immunized and stressed simultaneously (p?     

Inhibitory effect of 1alpha,25-dihydroxyvitamin D3 on allogeneic lymphocyte stimulation and Langerhans cell maturation

1alpha,25-Dihydroxyvitamin D3 (calcitriol) has been shown to inhibit the proliferation of peripheral blood mononuclear cells induced by allogeneic Langerhans cells in a human mixed epidermal cell lymphocyte reaction. The potent antigen-presenting function of Langerhans cells is gained during culture. We tried to dissect the effect of calcitriol on lymphocyte proliferation and Langerhans cell maturation in a murine mixed epidermal cell lymphocyte reaction using unfractioned epidermal cells as a source for Langerhans cells. First, calcitriol was added upon setup of the mixed epidermal cell lymphocyte reaction using cultured epidermal cells as antigen-presenting cells, and this was found to inhibit the proliferation of lymphocytes in a time- and concentration-dependent manner. When calcitriol was added only during preculture of freshly isolated epidermal cells, the subsequent mixed epidermal cell lymphocyte reaction was also inhibited. In addition, the growth of keratinocytes and the production of granulocyte/macrophage colony-stimulating factor during preculture of epidermal cells was completely inhibited. Supplementation with this growth factor only partially restored the proliferative response of lymphocytes. These results suggest that calcitriol inhibits both the alloantigen-driven stimulation of naive T cell and Langerhans cells maturation. Further experiments with purified Langerhans cells are required to elucidate the mechanism of action of calcitriol on Langerhans cell maturation.     

p40 molecule regulates NK cell activation mediated by NK receptors for HLA class I antigens and TCR-mediated triggering of T lymphocytes

p40 was previously described as a regulatory molecule capable of inhibiting both the natural and the CD16-mediated cytotoxicity of NK cells. In this study, we analyze the effect of p40 molecule engagement on the NK cell triggering induced by activating HLA class I-specific NK receptors (NKR) or on TCR alpha beta-mediated T cell activation. CD3-CD16+ NK cell clones expressing activating NKR (either CD94 or p50) were analyzed in a redirected killing assay using P815 target cells and appropriate mAb. A strong target cell lysis was detected in the presence of anti-NKR or anti-CD16 mAb alone. Addition of anti-p40 mAb resulted in a strong inhibition of both anti-NKR or anti-CD16 mAb-induced cytolysis. mAb specific for either CD45 or lymphocyte function associated antigen-1 did not exert any inhibitory effect in the same experimental system. Free intracellular calcium ([Ca2+]i) increase induced by mAb cross-linking of activating CD94 or p50 was inhibited by simultaneous engagement of p40 molecules, but not of other NK surface molecules including CD44 and CD56. In addition, cross-linking of p40 molecules strongly inhibited the CD94-induced tumor necrosis factor-alpha and IFN-gamma production. Analysis of TCR alpha beta or gamma delta T cell clones revealed that the engagement of p40 molecules, using specific mAb, induced some degree of inhibition only on anti-V beta (but not anti-V delta or anti-CD3) mAb-induced cytotoxicity. On the other hand, the p40 molecule engagement prevented T cell proliferation induced by either anti-V beta 8 or anti-V delta 2 mAb. A similar inhibitory effect was found on the IL-2-induced NK cell proliferation. Taken together, our present findings suggest that p40 may play a role in the regulation of NK and T lymphocyte activation and proliferation.     

Hypotensive action of bromocriptine in the DOCA-salt hypertensive rat: contribution of spinal dopamine receptors

Clinical islet transplantation is potentially the treatment of choice for people with type I diabetes. Rates of insulin independence in islet transplant recipients are disappointingly low, and the relative contribution of the rejection response compared with the loss of islet function is still unclear. We have compared the mixed lymphocyte islet coculture (MLIC) with the mixed lymphocyte acinar cell coculture (MLAC) and the mixed lymphocyte response (MLR) as in vitro models of allograft rejection to MHC and tissue-specific antigens expressed by human islets and acinar cells. The reduced number of MHC class II antigen-positive cells in islets and acinar tissue compared to those in the stimulator lymphocyte population of the MLR, correlated with a reduced proliferative response in the MLIC and MLAC. Enhancement of MHC class II antigen expression by islets using TNFalpha and IFNgamma did not increase their stimulatory capacity in the islet cocultures, which may have been due to a corresponding absence of B7 expression. The lack of T cell proliferation to acinar cells despite cytokine-induced enhancement of MHC class II expression and detectable B7 expression appeared to be due to the inhibitory effect of exocrine enzymes on lymphocyte proliferation. In conclusion, we suggest that a rejection response to islets and acinar tissue is possible due to the accompanying MHC class II-positive cells and that, in this model, islet and acinar-specific antigens do not significantly contribute to that response. Acinar cells may have the potential to stimulate lymphocytes directly, but this was not evident by proliferation in the MLAC. Rejection appears to contribute to the low survival rate of human islet allografts, but it is unlikely that this is the sole explanation, and other factors should be considered.     

Monocytes control gamma/delta T-cell responses by a secreted product

Gamma-irradiated ex vivo bovine monocytes induce proliferation of gamma/delta T cells in the autologous mixed lymphocyte reaction (AMLR), whereas when not irradiated they prevent this response. In contrast, non-irradiated autologous monocytes have no effect on bovine alpha/beta T-cell proliferation in the allogenic MLR suggesting that the regulation is specific for gamma/delta T-cell responses. Here, we showed that the inhibition was not mediated by inducing cell death and that the ability of ex vivo monocytes to prevent proliferation of gamma/delta T cells was not generalized in that gamma/delta T cells still responded to mitogenic stimulation. Inhibition of the AMLR by non-irradiated monocytes could not be overcome by addition of interleukin-2 to the cultures or by costimulation with antibodies to WC1, a gamma/delta T-cell-specific cell-surface differentiation antigen shown elsewhere by us to be involved in activation of gamma/delta T cells. Furthermore, we showed that monocytes inhibited gamma/delta T-cell responses via a soluble product since inhibition occurred even when monocytes and gamma/delta T cells were separated by membranes of transwells or when supernatants from monocyte cultures were added to AMLR cultures. Maximal secretion of the inhibitory product by the monocytes occurred during the first 6 hr of in vitro culture at 37 degrees, rapidly decreased thereafter, and did not occur when monocytes were incubated at 4 degrees. The inhibition was not attributable to nitric oxide, reactive oxygen intermediates, prostaglandin E2 or transforming growth factor-beta (TGF-beta) but the ability of monocyte supernatants to mediate inhibition was sensitive to heating at 65 degrees. Lipopolysaccharide and granulocyte-macrophage colony-stimulating factor activation of monocytes temporarily abrogated their ability to inhibit proliferation. In contrast, heat-shocking had no effect on their ability to inhibit. We hypothesize that non-irradiated monocytes produce the inhibitory material in vivo in order to regulate gamma/delta T-cell responses to self-derived monocyte membrane components, but that when monocytes are altered by infection, transformation, irradiation, or cytokine activation, production of the inhibitor is temporarily suspended allowing stimulation of gamma/delta T cells to occur.     

Human interleukin-10 can directly inhibit T-cell growth

Human interleukin-10 (IL-10) inhibits T-cell proliferation and cytokine production in the presence of monocytes. In this study, we have investigated whether IL-10 can directly inhibit T cells. Highly purified peripheral blood T cells containing less than 0.1% CD14+ cells and unresponsive to phytohemagglutinin (PHA), were growth-inhibited by IL-10 when stimulated with immobilized OKT3 monoclonal antibody (MoAb; 55.4% inhibition). This effect was neutralized by the murine MoAb 19F1 directed against human IL-10. In addition, IL-10 inhibited by 52.5% the proliferation of a human tetanus toxoid-specific T-cell clone (TM11) induced by immobilized OKT3 MoAb in the absence of antigen-presenting function. T-cell growth inhibition by IL-10 did not reflect a cytokine-induced change in the kinetics of T-cell response to immobilized OKT3 MoAb, and was observed over a wide range of cell and OKT3 MoAb concentrations. Addition of 1% to 5% monocytes to highly purified peripheral blood T cells resulted in the emergence of proliferation to PHA and to soluble OKT3 MoAb, but did not significantly affect levels of growth inhibition by IL-10 in the presence of immobilized OKT3 MoAb. Similarly, addition of 10% monocytes to the TM11 T-cell clone resulted in the emergence of proliferation in response to tetanus toxoid, but did not significantly influence growth inhibition by IL-10 in the presence of immobilized OKT3 MoAb. When stimulated with immobilized OKT3 MoAb in the absence of accessory cells, T cells secreted IL-2. Secretion of IL-2 under these conditions was inhibited by IL-10 (51.5% inhibition). Thus, IL-10 can directly inhibit growth and IL-2 production in T cells triggered by immobilized OKT3 MoAb in the absence of monocytes.     

High-throughput screening: advances in assay technologies

The expression and up-regulation of cell adhesion molecules on a human colonic epithelial cell line HT-29, and the peripheral blood T lymphocyte proliferation responses to bacterial superantigens presented by this cell line were investigated, compared with peripheral blood monocytes. In HT-29 cells, there was constitutive expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) at a low level, but no constitutive expression of HLA-DR, LFA-1, B7-1 and B7-2 molecules. After stimulation with the supernatants of staphylococcal enterotoxin B (SEB)-stimulated peripheral blood mononuclear cells for 48 h, there was significant up-regulation of HLA-DR and ICAM-1 molecules (both > 90% positive). However, this stimulation had no effect on the expression of LFA-1, B7-1, B7-2 and LFA-3 molecules. In the presence of all tested superantigens SEB, toxic shock syndrome toxin-1, and streptococcal pyogenic exotoxin A, stimulated HT-29 cells caused significant T cell proliferation. When monocytes were used as antigen-presenting cells (APC), the MoAbs against HLA-DR, B7-2 and LFA-3 showed a significant inhibition of SEB-induced T cell proliferation. Anti-ICAM-1 MoAb had no effect on this response. On the other hand, when stimulated HT-29 cells were used as APC, the MoAbs against HLA-DR and ICAM-1 significantly inhibited SEB-induced T cell proliferation. In contrast to monocytes, anti-B7-2 and anti-LFA-3 had no effect on this response. SEB could not induce HT-29 cells to produce IL-8 directly; however, SEB significantly induced the stimulated HT-29 cells to produce IL-8 in the presence of T cells. Thus these data demonstrate that the products of superantigen-stimulated T cell activation can increase the expression of HLA-DR and ICAM-1 molecules on HT-29 cells significantly. Stimulated HT-29 cells can serve as APC to bacterial superantigens. This response is an HLA-DR- and ICAM-1-dependent, but B7-2- and LFA-3-independent process, which was different from professional APC monocytes.     

Use of a whole blood assay to monitor the immune response to mycobacterial antigens in leprosy patients: a predictor for type 1 reaction onset?

In this study, we investigated tick saliva effects on T cell proliferation, antigen presentation and IFN-gamma-induced macrophage activation, events which are associated with host immune defense mechanisms. Mice repeatedly infested with Rhipicephalus sanguineus ticks, similarly to dogs, did not develop resistance to further infestations. We determined that R. sanguineus tick saliva inhibited, in a dose-dependent manner, both Con-A and specific antigen-induced splenic T cell proliferation. Tick saliva diluted twenty times (64 microg/ml) inhibited Con-A-induced and antigen-specific T cell proliferation in 83% and 69%, respectively. In addition, the inhibition of cell proliferation correlated with a decrease in IL-2 production. Microconcentrator fractionated saliva was tested on a Con-A-induced cell proliferation assay, and showed that one fraction between 3 and 10 kDa and another smaller than 3 kDa can be responsible for the inhibition of T cell proliferation. Although saliva inhibited cell proliferation, it did not impair antigen presentation. Tick saliva further abrogated the killing of intracellular forms of Trypanosoma cruzi by IFN-gamma-activated macrophages. Moreover, saliva-induced macrophage inhibition of IFN-gamma-induced-trypanocidal activity was paralleled with 69% less nitric oxide (NO) production. Finally, tick saliva doubled the production of IL-10 and reduced 84.6% production of IFN-gamma by splenocytes cultured with T. cruzi, suggesting that decreased macrophage NO production may be due to a saliva-induced cytokine imbalance, leading to decreased NO synthase activity. Together, these data indicate that tick saliva can modulate host immune response, thus, contributing to its feeding success and favoring the transmission of tick-borne pathogens.     

Inhibition of lymphocyte proliferation by factor(s) produced by Schistosoma mansoni

Normal spleen cells of CBA mice or Fischer rats were cultured with mitogens or allogeneic cells, together with various substances of Schistosoma mansoni origin, and thymidine uptake was measured. The proliferation (DNA synthesis) of normal lymphocytes was inhibited by the incubation product of the parasite as well as by cell-free supernatant of schistosome culture. Inhibition was obtained only when active materials were added at the beginning of the culture. Both T and B cell proliferation were inhibited. The inhibitory activity found in cell-free supernatant suggested the release by the parasite of some factor(s) interfering with lymphocyte proliferation. Moreover, serum from rats infected by S. mansoni inhibited lymphocyte proliferation also. The inhibitor(s) appeared heat resistant, dialyzable and of low molecular weight (500-1000). Incubation of normal spleen cells with S. mansoni inhibitor(s) did not enhance the release of nonspecific suppressor cell factor. The inhibition of product(s) released by the parasite could explain part of the immunosuppression status found in schistosomiasis.     

Effect of dexamethasone on cell proliferation of neuroepithelial tumor cell lines

The effect of glucocorticoid on cell proliferation, the expression of glucocorticoid receptor, and the relationship between inhibition of cell growth and apoptosis were investigated in four established neuroepithelial tumor cell lines (KNS42, T98G, A172, and U251MG). Glucocorticoid receptor expression was located in the cytoplasm of untreated cells, but translocated into nuclei after treatment with dexamethasone in KNS42, T98G, and A172 cells. U251MG did not express glucocorticoid receptors. Dexamethasone significantly inhibited the growth of KNS42 and T98G cell lines, at high concentrations in contrast to growth stimulation at low concentration. Dexamethasone inhibited proliferation of A172 cell line at all concentrations from 10(-4) M to 10(-7) M. These were prevented by RU38486, a specific glucocorticoid antagonist. Apoptosis did not occur in any cell lines after dexamethasone treatment. There was no response to glucocorticoid by U251MG cells. Dexamethasone treatment of neuroepithelial tumor cells expressing glucocorticoid receptors causes translocation into the nucleus to modulate cell proliferation upon binding of different concentrations of dexamethasone in vitro. Dexamethasone inhibits proliferation of some neuroepithelial cell lines, not by glucocorticoid-induced apoptosis. The bimodal potential of glucocorticoid to stimulate or suppress proliferation of neuroepithelial tumor cells expressing glucocorticoid receptor must be considered in clinical trials.