Mg2+ coordination in catalytic sites of F1-ATPase

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TNF-alpha), IL-1alpha, or IL-1beta. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-alpha stimulated IL-6 production by HGF, > 10-fold-larger amounts were induced with IL-1alpha and IL-1beta. Furthermore, the addition of both IL-1alpha and TNF-alpha to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-alpha, 1,000-fold by IL-1alpha and IL-1beta, and 1,400-fold by IL-1alpha plus TNF-alpha. IL-1alpha and TNF-alpha alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1alpha and TNF-alpha to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.     

Sleep apnea in end stage renal disease

Antibodies directed against the beta chain of the T-cell receptor (TCR) have been detected in animals and in humans in a number of distinct immune states that do not involve direct immunization with either T cells or TCR epitopes. When C57B1/6 mice are infected experimentally with the LP-BM5 retrovirus mixture they produce increased titres of autoantibodies directed against TCR V beta complementarity determining region 1 (CDR1) epitopes. Here, the authors utilized hybridoma technology to isolate monoclonal immunoglobulin (Ig)M antibodies (MoAbs) that arose at the peak of infection. The authors characterized the binding specificity tested using synthetic peptides modelling the CDR1 segments of 24 distinct V beta gene products and determined the VH gene usage by two such monoclonals. One binds to a restricted set of TCR V beta CDR1 peptides, and the second reacts with approximately half of the CDR1 peptide homologues. These MoAbs are specific for T-cell receptor beta chains and do not bind to immunoglobulin light chains or to unrelated protein molecules. Both MoAbs bind to intact T cells expressing the V beta domain (human V beta 8 and mouse V beta 11) from which selection peptides were derived, and costimulate a V beta specific in vitro T cell proliferative response induced by the staphylcoccal enterotoxin E (SEE) superantigen.     

Two separate mechanisms of T cell clonal anergy to Mls-1

T cell tolerance to superantigen can be mediated by clonal anergy in which Ag-specific mature T cells are physically present but are not able to mount an immune response. We induced T cell unresponsiveness to minor lymphocyte stimulations locus antigen (Mls)-1a in mice transgenic for TCR V beta 8.1 in three different systems: 1) injection of Mls-1a spleen cells, 2) mating with Mls-1a mice, and 3) bone marrow (BM) chimeras in which Mls-1a is present only on nonhematopoietic cells. CD4+8-V beta 8.1+ cells from all these groups did not proliferate in response to irradiated spleen cells from Mls-1a mice. We compared the response of these cells by T cell/stimulator cell conjugate formation, Ca2+ mobilization, and proliferation assays. The mechanisms underlying the unresponsiveness of these T cells appear to differ. CD4+8-V beta 8.1+ cells from Mls-1a spleen cell-injected mice mobilized cytoplasmic Ca2+ but proliferated at a reduced level in response to cross-linking with anti-TCR mAb. However, these cells formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a when activated B cells were used as stimulators, although they produced reduced levels of IL-2. In Mls-1a/b V beta 8.1 transgenic mice, a subset in CD4+8-V beta 8.1+ cells did not mobilize cytoplasmic Ca2+ after TCR cross-linking. Their conjugate formation, Ca2+ mobilization, or proliferation in response to Mls-1a on activated B cells was undetectable. Finally, CD4+8-V beta 8.1+ cells from the BM chimeras proliferated to TCR cross-linking at a partially reduced level and formed conjugates, mobilized Ca2+, and proliferated in response to Mls-1a on activated B cells. These features suggest that the mechanisms underlying the maintenance of anergy in Mls-1a spleen cell-injected mice are distinct from those in Mls-1a mice.     

Interleukin-6 production in human fibroblasts derived from periodontal tissues is differentially regulated by cytokines and a glucocorticoid

Interleukin-6 (IL-6) is thought to be a major mediator of the host's defense against infection, and it regulates immune responses in inflamed tissue. In this study, we investigated the regulation of IL-6 production in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). Pro-inflammatory cytokines including interleukin (IL)-1 alpha, IL-1 beta and tumor necrosis factor (TNF)-alpha stimulated IL-6 production in HGF and HPLF in a time- and dose-dependent manner. This IL-1 alpha, IL-1 beta, or TNF-alpha-induced IL-6 production was enhanced, but the cAMP accumulation they induced was inhibited by the addition of indomethacin. This result suggests that endogenous prostaglandin E2 (PGE2) partially inhibits IL-1 or TNF-alpha-induced IL-6 production and that the enhancement of IL-6 production by IL-1 or TNF-alpha may not be caused through endogenous PGE2-induced cAMP-dependent pathway. Dexamethasone (DEX), a glucocorticoid which is a inhibitor of nuclear factor kappa B (NF-kappa B activation, markedly inhibited IL-1 (alpha or beta) or TNF-alpha-induced IL-6 production; so this production may be partially mediated through NF-kappa B. IL-1 (alpha or beta) and TNF-alpha enhanced IL-6 production synergistically. IL-6 production in HGF or HPLF stimulated with IL-1 beta was augmented by the addition of interferon (IFN)-gamma, but was slightly suppressed by the addition of IL-4. Endogenous IL-6 enhanced IL-1 (alpha or beta)-induced IL-6 production in the presence of IL-6 soluble receptor (IL-6sR). Accordingly, in inflamed periodontal tissues, gingival fibroblasts and periodontal ligament fibroblasts stimulated with pro-inflammatory cytokines such as IL-1 or TNF-alpha, may produce IL-6, and this production can be differentially modulated by endogenous PGE2, IL-6sR, T cell-derived cytokines such as IFN-gamma or IL-4, and glucocorticoids.     

In vivo expression of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumour necrosis factor-alpha and interferon-gamma in the fetal murine thymus

Cytokines are known to play a role in T-cell lymphopoiesis as potent growth or differentiation factors, but many experiments focusing on their role in the thymus have been conducted only in vitro. We have thus used frozen sections obtained from fetal thymuses of normal C57BL 6 mice to investigate by immunohistochemistry the presence of interleukin-1 beta (I4-1 beta), IL-2. IL-4. IL-6. interferon-7 (IFN-7) and tumour necrosis facor-alpha (TNF-alpha). The results reveal that apart from IL-2, which was not detected, all these cytokines display a time-dependent expression pattern in the normal fetal thymus. First, production of IL-4, IL-6 and TNF-alpha is detected around days 13 14; this is followed by a second wave on days 16 17, with a production of IL-1 beta, IL-4 and IL-6, and finally, just before birth (day 19), by a third wave of IL-1 beta, IL-4, IL-6, IFN-7 and TNF-alpha production. This supports the hypothesis that cytokines play a rote in T-cell lymphopoiesis.     

A subset of CD4+ T cells expressing early activation antigen CD69 in murine lupus: possible abnormal regulatory role for cytokine imbalance

Systemic lupus erythematosus (SLE), which spontaneously develops in (NZB (New Zealand Black) x NZW (New Zealand White)) F1 mice, is strictly dependent on CD4+ T cells. We found that in these mice with overt SLE, CD4+ T cells expressing CD69 molecules, an early activation Ag, are dramatically increased in peripheral lymphoid tissues and inflammatory infiltrates in the kidney and lung, but not in peripheral blood, while CD8+ and NK1.1+ T cells were virtually CD69-. Various adhesion molecules, including LFA-1, ICAM-1, CD43, CD44, P-selectin, and E-selectin, were up-regulated. Analysis of the TCR repertoire showed no skewed TCR Vbeta usage. Studies on in vitro cytokine production of spleen cells on TCR cross-linking indicated that compared with findings in young mice, the aged mice showed severely impaired production of IL-2, IL-3, and IL-4, whereas the levels of IL-10 and IFN-gamma remained relatively intact. FACS-sorted CD69-CD4+ T cells from aged mice produced substantial amounts of these cytokines, including IL-2, IL-3, and IL-4, whereas CD69+CD4+ T cells were poor producers. Intriguingly, when cocultured, CD69+CD4+ T cells significantly inhibited the production of IL-2 by CD69-CD4+ T cells. IL-2 production by spleen cells from young mice was also markedly inhibited in the presence of CD69+CD4+ T cells obtained from aged mice. We propose that CD69+CD4+ T cells that are continuously activated by self peptides bound to MHC class II molecules in (NZB x NZW)F1 mice may be involved in the pathogenesis of SLE through abnormal regulatory effects on cytokine balance.     

Pattern of cytokines and pharmacomodulation in sepsis induced by cecal ligation and puncture compared with that induced by endotoxin

The production of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 and their pharmacomodulation were evaluated in a model of polymicrobial sepsis induced in mice by cecal ligation and puncture (CLP) and were compared with the effects of endotoxin (lipopolysaccharide [LPS]) treatment. LPS levels rose as early as 1 h after CLP and increased further after 2 and 21 h. TNF-alpha was detectable in serum, spleen, liver, and lungs during the first 4 h, with a peak 2 h after CLP. IL-1 beta was measurable in serum after 24 h, and levels increased significantly in spleen and liver 4 and 8 h after CLP. IL-6 levels increased significantly in serum throughout the first 16 h after CLP. These cytokines were detectable after LPS injection, with kinetics similar to those after CLP but at a significantly higher level. To cast more light on the differences between these two animal models of septic shock, we studied the effects of different reference drugs. Pretreatment with dexamethasone (DEX); ibuprofen (IBU), an inhibitor of cyclooxygenase; and NG-nitro-L-arginine, an inhibitor of nitric oxide synthase, significantly reduced survival, while chlorpromazine (CPZ) and TNF did not affect it. Only the antibiotics and pentoxifylline significantly increased survival in mice with CLP. However, CPZ and DEX protected the mice from LPS mortality. On inhibiting TNF-alpha with DEX, CPZ, or pentoxifylline, survival was reduced, unchanged, and increased, respectively, and on increasing TNF-alpha with IBU and TNF, survival was decreased or unchanged, respectively, suggesting that the modulation of this cytokine does not play a significant role in sepsis induced by CLP, unlike treatment with LPS. The negative effects of IBU and N(G)-nitro-L-arginine suggest a protective role by prostaglandins and nitric oxide in sepsis induced by CLP.     

Reduction of fecal contamination of street-vended beverages in Guatemala by a simple system for water purification and storage, handwashing, and beverage storage

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate-bound anti-CD3 monoclonal antibody (mAb) alone or also by soluble anti-CD28 mAb. Supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti-CD3 alone is not sufficient to induce interleukin-2 (IL-2) production in CD4+ cells from both young and old mice. However, anti-CD28, together with anti-CD3 mAb, induces a much higher production of IL-2 in CD4+ cells from young as compared with old mice. Conversely, interferon-gamma (IFN-gamma) production is also induced by anti-CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti-CD28 mAb, IFN-gamma production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL-4 and IL-10 is induced by anti-CD3 mAb but it is increased by the addition of anti-CD28 mAb. CD4+ cells from old mice produce more IL-4 and IL-10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti-CD3 or costimulated also by anti-CD28 mAb, the IFN-gamma, IL-4 and IL-10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.     

Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice

Con A-induced hepatic injury of mice accompanied by elevated transaminase was inhibited after in vivo depletion of liver NK cells and NK1+ T cells with intermediate TCR by anti-NK1 Ab or anti-IL-2Rbeta Ab. However, depletion of liver NK cells alone by anti-asialo-GM1 Ab did not inhibit hepatic injury. Although depletion of NK1+ T cells inhibited Con A-induced IL-2R expression of CD4+ high TCR (TCRhigh) cells and IL-4 mRNA expression of hepatic mononuclear cells, exogenous IL-4 engendered Con A-induced hepatic injury and endowed the expression of IL-2R of CD4+ TCRhigh cells. It was also found that in vivo treatment with anti-IL-4 Ab before Con A administration inhibited Con A-induced hepatic injury. In addition, although Con A did not induce hepatic injury in MHC class I-deficient mice, exogenous IL-4 again engendered severe hepatic injury in these mice. Further, while serum TNF-alpha levels induced by Con A were greatly decreased in NK1+ T cell-depleted mice and class I-deficient mice, TNF-alpha levels were recovered by exogenous IL-4. These findings reveal that although CD4+ TCRhigh cells in the liver and their production of TNF-alpha are the direct effectors of Con A-induced hepatic injury, liver NK1+ T cells also play an important role in this hepatitis model. Con A hepatitis may serve as an experimental model for human autoimmune hepatitis.     

Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and cytokines

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-alpha from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-alpha+ and most were also IL-6(+). Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-alpha and/or IL-6. IL-4(+) cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2(+) and/or IFN-gamma+ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-alpha and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.     

The effect of TCR V beta 8 peptide protection and therapy on T cell populations isolated from the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

Vaccination or treatment of Lewis rats with TCR V beta 8 peptides can prevent or reverse the clinical signs of experimental autoimmune encephalomyelitis (EAE) which is mediated predominantly by V beta 8.2+ CD4+/CD45R lo T cells. However, rats protected or treated with V beta 8 peptides still developed histological lesions in the spinal cord (SC), even though they remained clinically well. We sought to discern phenotypic changes characteristic of these SC infiltrating lymphocytes. In particular, we focused on whether the immunoregulatory mechanism induced by TCR peptides caused a reduction of V beta 8.2+ T cells, or induced changes in CD45R lo or hi/CD4+ subpopulations that have been associated respectively with EAE induction or recovery. In the V beta 8 peptide vaccinated rats there was a dramatic decrease in the number of V beta 8.2+ T cells isolated from the SC early in disease. During the recovery phase, however, the number of V beta 8.2+ SC T cells was similar in protected and control groups; in contrast, there was striking reduction in the number and size of CD45R hi/CD4+ T cells in the protected animals. In rats treated with V beta 8.2 peptide, no changes were observed in the number of SC V beta 8.2+ T cells or expression of V beta 8.2 message, but similar to vaccinated rats, there was a marked decrease in the number of CD45R hi/CD4+ T cells. These data suggest that vaccination with TCR peptides prevented the initial influx of encephalitogenic V beta 8.2+ T cells into the central nervous system (CNS), whereas treatment appeared to inactivate V beta 8.2+ T cells already present in the CNS. In both cases, TCR peptide-induced inhibition of the encephalitogenic T cells apparently preempted the need for CD45R hi/CD4+ T cells that may normally be necessary to resolve the disease.     

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.     

Flow-microfluorometric monitoring of oligoclonal CD8+ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo

The TCR repertoire of CD8+ T cells specific for Moloney murine leukemia virus (M-MuLV)-associated Ags has been investigated in vitro and in vivo. Analysis of a large panel of established CD8+ CTL clones specific for M-MuLV indicated an overwhelming bias for V beta4 in BALB/c mice and for V beta5.2 in C57BL/6 mice. These V beta biases were already detectable in mixed lymphocyte:tumor cell cultures established from virus-immune spleen cells. Furthermore, direct ex vivo analysis of PBL from BALB/c or C57BL/6 mice immunized with syngeneic M-MuLV-infected tumor cells revealed a dramatic increase in CD8+ cells expressing V beta4 or V beta5.2, respectively. M-MuLV-specific CD8+ cells with an activated (CD62L-) phenotype persisted in blood of immunized mice for at least 2 mo, and exhibited decreased TCR and CD8 levels compared with their naive counterparts. In C57BL/6 mice, most M-MuLV-specific CD8+ CTL clones and immune PBL coexpressed V alpha3.2 in association with V beta5.2. Moreover, these V beta5.2+ V alpha3.2+ cells were shown to recognize the recently described H-2Db-restricted epitope (CCLCLTVFL) encoded in the leader sequence of the M-MuLV gag polyprotein. Collectively, our data demonstrate a highly restricted TCR repertoire in the CD8+ T cell response to M-MuLV-associated Ags in vivo, and suggest the potential utility of flow-microfluorometric analysis of V beta and V alpha expression in the diagnosis and monitoring of viral infections.     

CD4+ T-cell population mediates development of inflammatory bowel disease in T-cell receptor alpha chain-deficient mice

BACKGROUND & AIMS: Increase of T cells expressing CD4 and T-cell receptor (TCR) alpha- beta+ (beta[dim]) was observed in the mucosal and peripheral lymphoid tissues of TCR alpha-/- mice with inflammatory bowel disease (IBD). The aim of this study was to characterize the CD4+ TCR alpha-beta+ T cells. METHODS: Cytokine production, TCR V beta usage, and helper function for Peyer's patch B cells by the CD4+ TCR alpha-beta+ T cells were assessed. RESULTS: The CD4+ TCR alpha-beta+ T cells purified from mesenteric lymph nodes and lamina propria of the intestine of IBD mice exclusively produced interleukin 4, used selected subsets (V beta6, V beta8, V beta14, and V beta15) of TCR, and massively proliferated after stimulation with staphylococcal enterotoxin B. Addition of the CD4+ TCR alpha-beta+ T cells to Peyer's patch B-cell cultures markedly enhanced immunoglobulin (Ig) A, IgG, and IgM antibody responses. Furthermore, depletion of the TCR alpha-beta+ T cells with monoclonal antibody against TCR beta chain completely suppressed the onset of IBD and polyclonal B-cell activation in the TCR alpha-/- mice. CONCLUSIONS: These findings suggest the CD4+ TCR alpha-beta+ T cells-mediated development of IBD in TCR alpha-/- mice.