Neutral sphingomyelinase in physiological and measles virus induced T cell suppression.

T cell paralysis is a main feature of measles virus (MV) induced immunosuppression. MV contact mediated activation of sphingomyelinases was found to contribute to MV interference with T cell actin reorganization. The role of these enzymes in MV-induced inhibition of T cell activation remained equally undefined as their general role in regulating immune synapse (IS) activity which relies on spatiotemporal membrane patterning. Our study for the first time reveals that transient activation of the neutral sphingomyelinase 2 (NSM2) occurs in physiological co-stimulation of primary T cells where ceramide accumulation is confined to the lamellum (where also NSM2 can be detected) and excluded from IS areas of high actin turnover. Genetic ablation of the enzyme is associated with T cell hyper-responsiveness as revealed by actin dynamics, tyrosine phosphorylation, Ca2+-mobilization and expansion indicating that NSM2 acts to suppress overshooting T cell responses. In line with its suppressive activity, exaggerated, prolonged NSM2 activation as occurring in co-stimulated T cells following MV exposure was associated with aberrant compartmentalization of ceramides, loss of spreading responses, interference with accumulation of tyrosine phosphorylated protein species and expansion. Altogether, this study for the first time reveals a role of NSM2 in physiological T cell stimulation which is dampening and can be abused by a virus, which promotes enhanced and prolonged NSM2 activation to cause pathological T cell suppression.

MNSFß regulates placental development by conjugating IGF2BP2 to enhance trophoblast cell invasiveness.

OBJECTIVES: Success in pregnancy in mammals predominantly depends on a well-developed placenta. The differentiation of invasive trophoblasts is a fundamental process of placentation, the abnormalities of which are tightly associated with pregnancy disorders including preeclampsia (PE). Monoclonal nonspecific suppressor factor beta (MNSFß) is an immunosuppressive factor. Its conventional knockout in mice induced embryonic lethality, whereas the underlying mechanism of MNSFß in regulating placentation and pregnancy maintenance remains to be elucidated. METHODS: Trophoblast-specific knockout of MNSFß was generated using Cyp19-Cre mice. In situ hybridization (ISH), haematoxylin and eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) were performed to examine the distribution of MNSFß and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) at the foeto-maternal interface. The interaction and expression of MNSFß, IGF2BP2 and invasion-related molecules were detected by immunoprecipitation (IP), immunoblotting and quantitative real-time polymerase chain reaction (qRT-PCR). The cell invasion ability was measured by the Transwell insert assay. RESULTS: We found that deficiency of MNSFß in trophoblasts led to embryonic growth retardation by mid-gestation and subsequent foetal loss, primarily shown as apparently limited trophoblast invasion. In vitro experiments in human trophoblasts demonstrated that the conjugation of MNSFß with IGF2BP2 and thus the stabilization of IGF2BP2 essentially mediated the invasion-promoting effect of MNSFß. In the placentas from MNSFß-deficient mice and severe preeclamptic (PE) patients, downregulation of MNSFß was evidently associated with the repressed IGF2BP2 expression. CONCLUSIONS: The findings reveal the crucial role of MNSFß in governing the trophoblast invasion and therefore foetal development, and add novel hints to reveal the placental pathology of PE.

Sunitinib mediates reversal of myeloid-derived suppressor cell accumulation in renal cell carcinoma patients.

PURPOSE: Immune dysfunction reported in renal cell carcinoma (RCC) patients may contribute to tumor progression. Myeloid-derived suppressor cells (MDSC) represent one mechanism by which tumors induce T-cell suppression. Several factors pivotal to the accumulation of MDSC are targeted by the tyrosine kinase inhibitor, sunitinib. The effect of sunitinib on MDSC-mediated immunosuppression in RCC patients has been investigated. EXPERIMENTAL DESIGN: Patient peripheral blood levels of MDSC and regulatory T-cell (Treg) and T-cell production of IFN-gamma were evaluated before and after sunitinib treatment. Correlations between MDSC and Treg normalization as well as T-cell production of IFN-gamma were examined. The in vitro effect of sunitinib on patient MDSC was evaluated. RESULTS: Metastatic RCC patients had elevated levels of CD33(+)HLA-DR(-) and CD15(+)CD14(-) MDSC, and these were partially overlapping populations. Treatment with sunitinib resulted in significant reduction in MDSC measured by several criteria. Sunitinib-mediated reduction in MDSC was correlated with reversal of type 1 T-cell suppression, an effect that could be reproduced by the depletion of MDSC in vitro. MDSC reduction in response to sunitinib correlated with a reversal of CD3(+)CD4(+)CD25(hi)Foxp3(+) Treg cell elevation. No correlation existed between a change in tumor burden and a change in MDSC, Treg, or T-cell production of IFN-gamma. In vitro addition of sunitinib reduced MDSC viability and suppressive effect when used at >/=1.0 microg/mL. Sunitinib did not induce MDSC maturation in vitro. CONCLUSIONS: Sunitinib-based therapy has the potential to modulate antitumor immunity by reversing MDSC-mediated tumor-induced immunosuppression.

T-cell death and cancer immune tolerance.

Cancer patients mount adaptive immune responses against their tumors. However, tumor develops many mechanisms to evade effective immunosurveillance. T-cell death caused by tumor plays a critical role in establishing tumor immunotolerance. Chronic stimulation of T cells by tumors leads to activation-induced cell death. Abortive stimulation of T cells by tolerogenic antigen-presenting cells loaded with tumor antigens leads to autonomous death of tumor-specific T cells. Therapeutic approaches that prevent T-cell death in the tumor microenvironment and tumor draining lymph nodes, therefore, should boost adaptive immune responses against cancer.

Quercetin regulates the inhibitory effect of monoclonal non-specific suppressor factor beta on tumor necrosis factor-alpha production in LPS-stimulated macrophages.

Monoclonal non-specific suppressor factor beta (MNSFbeta) is a member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFbeta regulates the ERK1/2-MAPK cascade in the macrophage cell line Raw 264.7. In this study, we found evidence that the flavonol quercetin regulates the effect of MNSFbeta on TNFalpha production in LPS-stimulated Raw264.7 cells. Quercetin inhibited MNSFbeta siRNA-mediated enhancement of both TNFalpha production and ERK1/2 phosphorylation in LPS-stimulated Raw264.7 cells. Quercetin decreased the expression of 33.5-kDa MNSFbeta adduct, which is important to the regulation of ERK1/2 activity, in unstimulated Raw264.7 cells. The various flavonoids tested, including other flavonols, did not affect the formation of this adduct. Collectively, MNSFbeta and quercetin might share a common pathway in regulating the ERK1/2 pathway in macrophages. This is the first report describing the involvement of flavonoids in the action of ubiquitin-like proteins.

Inhibition of murine nephritogenic effector T cells by a clone-specific suppressor factor.

We have used a murine model of organ-specific autoimmunity to characterize therapeutic modalities capable of down-regulating the cellular limb of the autoimmune response. Murine interstitial nephritis is an autoimmune disease mediated by tubular antigen-specific CD8+ nephritogenic effector T cells which are delayed-type hypersensitivity (DTH) reactive and cytotoxic to renal epithelial cells. Previous studies have demonstrated that disease can be suppressed with experimentally induced populations of T cells (Ts1 and Ts2 cells) obtained after injection of tubular antigen-coupled splenocytes into syngeneic mice. As the target of Ts2 is the CD8+ effector T cell, we have evaluated its effects on nephritogenic effector T cell clones isolated from diseased animals. Our studies demonstrate that soluble proteins expressed by Ts2 cells (TsF2) specifically abrogate the DTH, cytotoxic, and nephritogenic potential of M52 cells, although T cell receptor and IL-2 receptor expression are unchanged in these unresponsive M52 clones. TsF2-induced inhibition is dependent on new mRNA and protein synthesis. In a cytotoxic clone, M52.26, exposure to TsF2 induces expression of TGF-beta 1 which is, in turn, required for inhibition of cytotoxicity and nephritogenicity. Our studies are consistent with TGF-beta 1 behaving, at least in some T cells, as a nonspecific final effector of clone-specific suppression.

T cell mediated immunosuppression.

Suppressor T cells down-regulate the activity of other cells in the immune system, and, albeit controversial, are believed to play a role in immunological tolerance and immunoregulation. Significant progress has been made in characterizing suppressor T cells and their receptors, and in elucidating mechanisms of immunosuppression. This knowledge is important for understanding the immune system and certain disease states and for favorably manipulating immunity.

Modification of host antitumor defense mechanisms in mice by progressively growing tumor.

The EL4 lymphoma in C57BL/6 mice was used as a model to examine the effect of progressive tumor growth on a variety of cell mediated cytolytic effector functions which have been shown in other systems to have antitumor potential. The functions examined were those of cytolytic T-lymphocyte, lymphokine activated killer cells, natural killer cells, and tumoricidal macrophage (MO). The kinetics of each function displayed a unique pattern as a consequence of tumor growth, but all were inhibited in animals bearing large tumors (late tumor bearers). In cell mixing experiments it was shown that spleen cells from individual late tumor bearers were suppressive for cytotoxic T-lymphocytes, lymphokine activated killer cells, and splenic MO but not peritoneal MO or splenic natural killer cells. The suppression was nonspecific and was mediated primarily by nonadherent cells and/or their soluble products. Suppression appeared to be mediated, in part, by tumor cells in the spleen since the degree of suppressor activity associated with a particular spleen cell preparation correlated with the number of tumor cells present. Furthermore, the direct addition of viable ascites EL4 cells to response cultures or assays had similar suppressive effects as late TBM spleen cells, i.e., inhibited cytotoxic T-lymphocytes, lymphokine activated killer cells, and splenic MO but had no effect on natural killer cells or peritoneal MO. The mechanism of suppression by ascites EL4 was not determined but it was mediated by viable cells only and not due to contaminating viruses or other microorganisms.

Role and function of antigen nonspecific suppressor factors.

Although antigen-nonspecific suppressor factors described by various investigators appear to exhibit a certain amount of heterogeneity in both physical and biological properties, these proteins also exhibit significant similarities. Nonspecific suppressor factors are generally produced by Ly 2+ (murine) or OKT8+ (human) T lymphocytes. One protein, soluble immune response suppressor (SIRS), is produced by T lymphocytes after incubation with mitogens, interferons, or histamine, and must be activated by peroxides to inhibit cell division or immune function. SIRS appears to inhibit cell division by causing oxidation of a portion of cellular protein sylfhydryls and, in particular, causes a decrease in intracellular levels of deoxyribonucleotide triphosphates. This decrease is readily reversed by sulhydryl reducing agents, such as 2-mercaptoethanol. The activity of SIRS and other suppressor factors is inhibited by growth factors, such as interleukin 2, and the activity of interleukin 2 is inhibited by antigen-nonspecific suppressor factors. Further, SIRS or SIRS-like proteins are produced during various diseases associated with suppressed immune responsiveness including acquired immune deficiency syndrome, schistosomiasis, and nephrotic syndrome. These data suggest that antigen-nonspecific suppressor factors may have an important physiological role in regulating immune responses and cell division in general.

Characterization of a monocyte differentiation factor distinct from gamma-interferon, tumor necrosis factor, or G,M-colony-stimulating factor that regulates the growth and functional capabilities of the U937 monocytic cell line.

We have analyzed the characteristics and cellular sources of the T cell-derived lymphokines that affect the proliferation and the oxidative metabolic capabilities of the U937 monocytic cell line. Although gamma-interferon (gIFN) and tumor necrosis factor-alpha (TNFa) can, in high doses, inhibit the proliferation of U937 cells, the predominant antiproliferative factor produced by activated CD4+ and CD8+ T cells has a MW = 45-55 Kd, is resistant to heat treatment, and is distinct and independent from gIFN, TNFa, and GM-CSF. The inhibitory effect of this lymphokine on U937 cell growth requires an 18-24-hr induction period; thereafter, this growth arrest persists for up to 5 d, even in the absence of the factor. The lymphokine responsible for inducing oxidative metabolic capabilities in U937 cells is also a non-gIFN, heat-resistant, 45-55-Kd factor secreted by CD4+ and CD8+ cells, and we postulate that this differentiation factor is identical to the factor responsible for inhibiting U937 growth. These data demonstrate the prominent role of T cell-derived factors distinct from gIFN, TNFa, or GM-CSF in regulating the growth and functional capabilities of monocyte-lineage cells. Furthermore, the data suggest it may be appropriate to distinguish monocyte activation, in which cells at a given maturational stage develop a heightened ability to perform a particular function, from changes in the functional repertoire of cells acquired as a consequence of lymphokine-induced differentiation.

Evidence for an epidermal cytokine network.

Cytokines are (glyco)proteins that are synthesized and secreted by various cells, which bind to specific receptors on target cells and which regulate activation, proliferation, and differentiation of immune as well as non-immune cells. Keratinocytes upon injury release interleukin (IL)-1, IL-6, IL-8, colony-stimulating factors, and tumor-necrosis factor, as well as growth and suppressor factors. There is also strong evidence for a network of interacting cytokines, which has been only partially characterized so far, maintaining a proper balance. However, excessive or insufficient production of these mediators may contribute to certain disease states, particularly those with infectious and autoimmune genesis. Therefore the understanding of cytokine interactions may be helpful in elucidating the pathomechanisms of such diseases. Moreover, certain cytokines, as well as their analogues and antagonists, may prove to be of therapeutic value.

The effects of perioperative portal venous inoculation with donor lymphocytes on renal allograft survival in the rat. I. Specific prolongation of donor grafts and suppressor factor in the serum.

In order to investigate the in vivo functional role of the liver in the immune responses in organ transplantation, effects of perioperative portal venous p.v. administration of donor lymphocytes on renal allograft survival were tested in the rat kidney transplant model. Donor lymphocytes were prepared from BN (BN, RT-1n) or third-party DA (RT1a) rat spleens and lymph nodes and injected p.v. or intravenously to Lewis (LEW, RT-1l) hosts on the day of transplantation (day 0). Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.6 days (n = 10). Intravenous administration of 1 x 10(8) BN cells to LEW hosts on day 0 caused a slight, but not significant, prolongation of renal allograft survival (MST = 9.5 +/- 3.0 days, n = 13, NS), whereas portal venous inoculation of 1 x 10(8) BN cells on day 0 remarkably prolonged renal graft survival to 22.2 +/- 5.3 (n = 10, P less than 0.01). The prolongation of graft survival was antigen-specific; the administration of 1 x 10(8) DA cells p.v. to LEW hosts did not prolong the survival of BN renal grafts (MST = 7.4 +/- 0.8, n = 5). Spleen cells from p.v. treated LEW hosts 10 days after transplantation had no suppressor effect on the one-way MLC reaction of normal LEW responder cells toward donor BN or third-party DA stimulators. On the other hand, when serum from p.v.-treated LEW hosts was added to MLC at a concentration of 3 per cent of total volume, it suppressed the MLC reaction toward donor BN cells by 71.6 per cent, but not toward third-party DA stimulators (-8.5 per cent suppression, NS). Histological examination of p.v.-treated LEW hosts at 10 days after transplantation revealed that the liver had normal lobular architecture without expansion of portal tracts and infiltration of inflammatory cells. On the other hand, the transplanted kidney demonstrated a moderate mononuclear cell infiltration around the artery without an interstitial hemorrhage. Moreover, adoptive transfer of the serum from p.v.-treated LEW rats into the virgin secondary LEW hosts significantly prolonged the graft survival of BN kidneys from 7.8 days to 18.9 +/- 5.5 days (P less than 0.01), but not third-party DA graft survivals (MST = 7.5 +/- 0.6 days), indicating that an antigen-specific tolerogenic factor was released into the circulation through the process of allogeneic cells in the liver.

Characterization and analysis of soluble suppressor factor from early human decidual cells.

To assess the role of decidual cells (DC) in the maintenance of pregnancy, immunosuppressive activity of culture supernatants from human DC were investigated. Dispersed DC suspensions from decidual tissue of early pregnancies were prepared by an enzyme digestion method using collagenase and DNase, and were enriched over 90 per cent without contamination of macrophages and lymphocytes in the fraction, with specific gravity between 1.033 and 1.044 (fraction 2 [Fr2] ) by a Percoll discontinuous density gradient method. The culture supernatants of Fr2 cells suppressed the responses of normal peripheral blood lymphocytes to PHA, MLR, and killer T cell generation at the 50 per cent concentration. To determine the mechanism of the immunosuppressive activity of the culture supernatants, the effect of the supernatants on interleukin-2 and gamma-interferon production, as well as IL-2 receptor expression, on PBL was investigated. The supernatants from 3 x 10(6)/ml of DC cells inhibited not only IL-2 and gamma-INF production, but also IL-2 receptor expression, compared with normal controls. The supernatants also suppressed immunoglobulin (IgG and IgM) production by pokeweed mitogen-stimulated B cells. To purify the suppressor factor from culture supernatants of DC, serum free culture supernatants of 3 x 10(6)/ml of DC, which showed 32 per cent of inhibitory activity on MLR, were applied to gel filtration. Fractions between mw 67,000 and 43,000 suppressed the MLR. These results suggest that DC from decidua of early pregnancy excrete an immunosuppressive factor with a molecular weight between 43,000 and 67,000 daltons.

Soluble suppressive molecule released by human T lymphocytes induced by heat-inactivated allostimulator cells in the presence of cyclosporine.

E-rosette-positive peripheral blood lymphocytes (E+PBL) stimulated initially with heat-inactivated allogeneic lymphoblastoid cells in the presence of cyclosporine (CsA/HI) produce a soluble molecule that suppresses fresh lymphocytes in a primary mixed lymphocyte reaction. Cell lines were derived from the E+PBL cells after one and two weeks of culture. These lines were CD4+ by both FACS and mRNA analysis. The cells produce a potent soluble molecule (supernates often containing greater than 1000 units of suppressive activity per milliliter). The factor has an apparent molecular weight of 90 k and is sensitive to both pH and boiling. The molecule is not the suppressive cytokine TGF beta, based on neutralization with anti-TGF beta antibody and mRNA expression. None of the available cytokines expressed by these cells was suppressive when titrated into an MLR, alone or in combination. These results support the conclusion that CsA/HI-activated T cell lines produce a novel cytokine that is not antigen-specific or MHC-restricted.

Human suppressor T cells induced in vitro with an autologous renal allograft-derived T cell line. II. Activity and specificity of a soluble suppressor factor.

Human suppressor T cells induced by autologous mixed lymphocyte reaction (AMLR) using fresh responder PBL from a renal transplant recipient and an autologous irradiated antidonor CTL line (EE-1) established from a biopsy of the patient's own allograft were studied for the production of suppressor factors. The suppressor cell lines propagated (designated TsEE) were capable of inhibiting the in vitro generation of proliferative and cytotoxic responses of responder cells from the recipient or other individuals who shared HLA-B7 with TsEE cells, regardless of the stimulatory cell phenotype. Coculture of TsEE cells with the autologous irradiated EE-1 inducer cell line in vitro yielded a soluble factor (designated TsEEF) capable of inhibiting the generation of MLR and CTL responses, as well as mitogen-induced proliferative responses to PHA and PWM in an HLA-unrestricted manner. TsEEF also inhibited the replication of lymphoblastoid T cell lines (Molt-4 and HSB) but not B cell lines (SB and JC-EBV) or PBL stimulated with the B cell mitogen LPS. Control supernatants obtained from each of the cells used to generate TsEE in AMLR (i.e., EE-PBL and the EE-1 line) cultured alone or together for 48 hr demonstrated no suppressive activity in any of these test systems. TsEEF was nontoxic for lymphoid cells, was nondialyzable (greater than 12kDa), did not act by interfering with IL-1 or IL-2 utilization, and was negative for TNF and IFN-gamma activity. Functionally, the suppressive activity of TsEEF was dose-dependent, did not shift MLR kinetics, and could be absorbed by T cells. T cells incubated with TsEEF for 4 hr were unresponsive to subsequent mitogen or MLR stimulation. These findings indicate that, whereas T suppressor cell lines propagated from the circulation of a stable renal transplant recipient demonstrate class I HLA restriction, the activity of their soluble products is not HLA-restricted, and functionally inhibits T cell proliferation.

Separation of allostimulatory and natural suppressor/stem cell functions of murine bone marrow--implications for bone marrow transplantation.

Natural suppressor (NS) activity is defined by the ability of a null cell population to inhibit antigenic and mitogenic activation of lymphoid cells. We had previously demonstrated that murine and human bone marrow was a rich source of NS activity and could be enriched in a population of large, low-density cells after counterflow centrifugal elutriation (CCE). In this report we confirm previous findings in the mixed lymphocyte reaction (MLR) that NS activity is an endogenous function of BM and is not present in peripheral immune tissues. Furthermore, suppression by BM NS cells in the MLR is not MHC restricted, which distinguishes NS activity from veto activity also associated with BM. To enrich for NS activity, BM cells were separated into 3 fractions according to size and density by CCE. Fraction (F) 1 contained small, high-density cells; F2 contained cells of intermediate size and density; and F3 comprised large, low-density cells. Addition of CCE-enriched fractions to the MLR revealed potent NS activity associated with F3, consistent with our previous findings in the Ab response. CCE-enriched fractions of BM cells were also used as stimulators in an MLR to determine which fraction, if any, could support alloantigen-induced proliferation. The use of unfractionated C57Bl/6 (H-2b) BM cells as stimulators for BALB/c (H-2d) splenic responders resulted in little detectable proliferation as compared with that induced by C57Bl/6 splenic stimulators. However, when elutriated fractions were used as stimulators, F1 BM cells induced a significant proliferative response, albeit to a lesser magnitude than spleen cells. In order to determine which fraction contained hematopoietic progenitors, CCE-separated cells were assayed for granulocyte/macrophage colony formation (CFU-GM). F3 BM was enriched five-fold for CFU-GM progenitors as compared with unfractionated BM, while progenitors were virtually absent in F1 and F2 BM. The results suggest that CCE may represent an effective way of removing potential alloantigen-presenting cells (F1) from BM, while retaining hematopoietic progenitors and NS activity (F3). The implications of the findings as they relate to allogeneic BM transplantation are discussed.

Glucocorticoid involvement in suppression of NK activity following surgery in rats.

We studied plasma factors mediating suppression of NK activity (NKA) following surgery. Plasma from operated rats suppressed NKA of splenocytes, leukocytes, and purified natural killer (NK) cells, and charcoal stripping nullified suppression. The glucocorticoid antagonist mifepristone prevented suppression, whereas blockers of reactive oxygen metabolites, opioids, catecholamines, prostaglandin-E2, and histamine did not. NKA dropped as corticosterone levels peaked postoperatively, and administration of relevant doses of corticosterone suppressed NKA. Inhibition of glucocorticoid synthesis prevented plasma from suppressing NKA but merely attenuated NKA suppression in operated rats. Thus, postoperative concentrations of corticosterone can directly suppress NKA but additional factors probably act in vivo.

Longevity of human allospecific TLCs: mycoplasma infection as a cause of in vitro "suppression" of MLC.

It has been suggested that allospecific T-cell clones lose specific reactivity after approximately 30 cell doublings and subsequently acquire suppressor and NK-like characteristics. We have tested this hypothesis by assaying paired functional and nonfunctional TLCs for suppressor activity in PLT and MLC cocultures. Two sets of clones were initially studied: the first pair consisted of clone S5.2B, a functional TLC, and S5.14A, a nonfunctional TLC; the second pair of clones tested was comprised of two different expansions of the same clone S5.5A (nonfunctional) and S5.5B (functional). These experiments yielded no evidence for suppressive activity by nonfunctional clones toward functional clones, furthermore, the addition of nonfunctional clones to primary MLC assays had no effect on the level of responsiveness. Eight clones were subcloned and 89 subclones were retested for function after approximately 50 cell doublings. Generally, the subclones failed to suppress MLC proliferation. A minority of TLCs could suppress MLC responses, but this "suppression" was reversible with the addition of 2% exogenous TCGF. However, eight subclones and two parental TLC lines did suppress MLC responses in the presence or absence of TCGF, but the suppressive effects in such cocultures were reversible in the presence of tylocine, an anti-mycoplasma antibiotic. Therefore, human T-cells, cultured for extended periods, do not inexorably and universally lose specific alloreactivity and gain suppressive characteristics due to some presumed differentiative event.

Bone marrow cells promote TH2 polarization and inhibit virus-specific CTL generation.

This laboratory recently reported that human bone marrow cells (BMC) inhibit the generation of virus-specific CTL in culture. The culture supernatants contained increased levels of prostaglandin E(2) (PGE(2)) (shown to favor TH2 cell development) and also inhibited EBV-CTL effector cell development. In this study, we obtained PBL from Epstein-Barr virus (EBV) IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) one year after renal transplantation. EBV-specific CTL were then generated in vitro by stimulating PBL with autologous EBV-transformed B cells (EBV-B) in the presence or absence of autologous BMC. The addition of BMC to the EBV-CTL generation cultures increased the intracellular expression in CD3+ cells of IL-4,-5,-6,-10, and -13. These CD3+ cells also expressed increased levels of the TH2 associated receptor CCR3. Inhibition was even observed by preparing EBV-CTL generating cultures in trans-wells that separated the autologous BMC from the PBL + EBV-B. It was then observed that CD3+ cells obtained after 7 days of culture in the presence of autologous BMC could be used as inhibitors of EBV-CTL generation. Protein Kinase A (PKA), a cAMP kinase that is involved in the upregulation of TH2 cytokine activity, was increased in EBV-CTL cultures by the presence of BMC. Additionally, IL-4-mediated signal transduction and activation of transcription (STAT-6) phosphorylation was slightly increased. These results show that the BMC inhibition is mediated by soluble factors (cytokines) and that cell-cell contact in this autologous system is not required, so that BMC (at least partially, via cytokine production) promote TH2 polarization in culture. Moreover, TH2 cells induced by culturing with autologous BMC directly inhibit EBV-CTL generation, and TH2 associated PKA, CCR3, and STAT-6 phosphorylation are enhanced by BMC.

Regeneration and tolerance factor modulates the effect of adenosine triphosphate-induced interleukin 1 beta secretion in human macrophages.

These studies characterize a molecule known as regeneration and tolerance factor (RTF), which controls inflammation by regulating interleukin 1 beta (IL-1 beta) secretion. Recently, it has been demonstrated that the interaction of adenosine triphosphate (ATP) with the P2X7 purinoceptor induces the secretion of IL-1 beta and initiates the inflammatory response. In these experiments, that the addition of ATP to macrophages was found to induce P2X7 activation and secretion of IL-1 beta. This secretion is enhanced with anti-RTF antibody in combination with exogenous ATP (p< 0.005). RTF is also revealed to be able to influence surface ATPase activity and, increase PI incorporation, which is an indicator of P2X7 activation. We demonstrate that RTF has a role in controlling IL-1 beta secretion by regulating P2X7 activity.