Effects of associated cytokines (IL-1, TNF-alpha, IFN-gamma and TGF-beta) on collagen and glycosaminoglycan production by cultured human synovial cells

The production of collagen and glycosaminoglycans (GAG) was studied in cultured human synovial cells exposed to four cytokines, alone or in dual combination, namely interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). Among these cytokines, only TGF-beta (0.1-10 ng/ml) induced a significant and dose-dependent increase of collagen synthesis in a 24-h incubation. This effect was reversed when the factor was associated with either IL-1 beta (100-500 pg/ml), TNF-alpha (1-100 ng/ml) or IFN-gamma (100 U/ml). Except IFN-gamma which clearly inhibits the collagen production, the other cytokines IL-1 and TNF-alpha were not very effective when tested separately, although they generally induced a small reduction in collagen amount. IL-1 beta and TNF-alpha were found to be more efficient than TGF-beta in stimulating the production of GAG by the synovial cells. IFN-gamma exerted an antagonistic effect on the TGF-beta-induced stimulation of GAG synthesis. TNF-alpha and IL-1 beta were shown to have an additive effect on that production. The results indicate that interactions between cytokines present in the inflamed synovial tissue may modulate their respective actions and thus introduce differentials in their effect on collagen and GAG metabolism which are responsible for the alterations of synovial extracellular matrix in rheumatoid arthritis.     

Augmented production of proinflammatory cytokines and accelerated allotransplantation reactions in heroin-treated mice

Heroin treatment or abusive drug addiction influences many physiological functions, including the reactions of the immune system. Although suppression of various manifestations of the immune system after heroin (or morphine) administration has been reported, we show here that production of proinflammatory cytokines and nitric oxide (NO) was enhanced and allotransplantation reactions were accelerated significantly in heroin-treated recipients. Mice were treated by a subcutaneous administration of heroin (diacetylmorphine) given in one or repeated daily doses. The ability of spleen cells from treated mice to respond in vitro to alloantigens and to produce IL-2, IL-4, IL-10 and IFN-gamma, and the production of IL-1beta, IL-12 and NO by peritoneal macrophages, were tested. Within 2 h after heroin administration, proliferative responses to alloantigens and the production of IL-1beta, IFN-gamma, IL-12 and NO were enhanced significantly. In contrast, the production of anti-inflammatory cytokines IL-4 and IL-10 was at the same time rather decreased. As a consequence, skin allografts in heroin-treated mice were rejected more promptly than in untreated or vehicle-treated recipients. Similarly, the growth of allogeneic tumours induced by high doses of tumour cells was suppressed significantly in heroin-treated mice. The enhancing effects of heroin on the production of proinflammatory cytokines were antagonized by naltrexone, a specific inhibitor of classic opioid receptors. These results show that heroin treatment augments production of proinflammatory cytokines and accelerates allotransplantation reactions. The observations thus illustrate the complexity of the effects of heroin on the immune system and should be taken into account during medical treatment of opiate addicts and in the use of morphine to decrease pain in various clinical situations.     

Synthesis and regulation of accessory/proinflammatory cytokines by intestinal epithelial cells.

Intestinal epithelial cells (IEC) have been shown to act as antigen-presenting cells (APC) in vitro and may have this capacity in vivo. In order to determine whether IEC, like other APC, are able to produce accessory cytokines which may play a role in T cell activation, we assessed the accessory cytokine profile of IEC constitutively or after stimulation. We measured expression, production and regulation of accessory cytokines (IL-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) by the presence of mRNA as well as secreted protein. Freshly isolated IEC from surgical specimens were cultured in the presence or absence of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), IL-1 beta or TNF-alpha. mRNA was assessed by a specific RNAse protection assay which controlled for contaminating cell populations while protein secretion was measured by ELISA (IL-1) or bioassay (TNF and IL-6). Neither IL-1 beta nor TNF-alpha were detectable in cultured IEC supernatants, supporting the lack of macrophage contamination. All IEC spontaneously secreted IL-6 at levels comparable to those of macrophages. IEC IL-6 mRNA also increased approximately 200-fold during the first 24 h of culture. LPS, IFN-gamma or TNF-alpha had no effect on spontaneous IL-6 production, and neither resulted in the secretion of IL-1 beta or TNF-alpha. However, IL-1 beta up-regulated IL-6 synthesis by 6-7-fold. IEC express a profile of cytokine mRNAs distinct from conventional APC (low level constitutive IL-6 expression but no detectable IL-1 beta, TGF-beta or TNF-alpha), adding to their uniqueness as APC.     

The role of tumour necrosis factor-alpha in combination with interferon-gamma or interleukin-1 in the induction of immunosuppressive macrophages because of Mycobacterium avium complex infection.

The role of some cytokines including tumour necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta) and interleukin-6 (IL-6) in the generation of immunosuppressive macrophages (M phi s) in host spleen cells of Mycobacterium avium complex (MAC)-infected mice was studied. M phi populations with potent suppressor activity against concanavalin A (Con A)-induced mitogenesis of splenocytes (SPCs) were elicited not only in euthymic but also in athymic nude mice during MAC infection. The suppressor M phi s are, therefore, inducible not only through a T-cell-dependent mechanism but also through T-cell-independent mechanism. However, MAC-induced M phi s of athymic mice displayed about four times lower suppressor activity than those of euthymic mice, indicating that mature T cells are important for M phi activation to the highly immunosuppressive state. Anti-TNF, anti-IFN-gamma, and anti-TGF-beta antibodies (Abs) but not anti-IL-6 Ab inhibited in vivo generation of MAC-induced immunosuppressive M phi s, and the neutralizing efficacy was in the order of anti-IFN-gamma Ab > anti-TNF Ab > anti-TGF-beta Ab. The effects of TNF-alpha, IL-1 alpha, IL-6, and IFN-gamma alone or combinations of them upon the acquisition of the suppressor activity by cultured splenic M phi s were studied. When normal splenic M phi s were treated with each cytokine for 3 days, TNF-alpha, IFN-gamma, and IL-1 alpha alone caused a slight elevation of their suppressive activity. Treatment of the normal M phi s with the combination of either TNF-alpha+IL-1 alpha or TNF-alpha+IFN-gamma yielded a marked increase in the suppressor activity, followed by IL-1 alpha+IFN-gamma. These findings indicate the important roles of TNF-alpha, IFN-gamma, and IL-1 alpha in the generation of MAC-induced suppressor M phi s.     

Interaction of Mycobacterium tuberculosis-Induced Transforming Growth Factor β1 and Interleukin-10

Mycobacterium tuberculosis is associated with the activation of cytokine circuits both at sites of active tuberculosis in vivo and in cultures of mononuclear cells stimulated by M. tuberculosis or its components in vitro. Interactive stimulatory and/or inhibitory pathways are established between cytokines, which may result in potentiation or attenuation of the effects of each molecule on T-cell responses. Here we examined the interaction of transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) in purified protein derivative (PPD)-stimulated human mononuclear cell cultures in vitro. TGF-beta1 induced monocyte IL-10 (but not tumor necrosis factor alpha) production (by 70-fold, P < 0.02) and mRNA expression in the absence but not in the presence of PPD. Both exogenous recombinant (r) IL-10 and rTGF-beta1 independently suppressed the production of PPD-induced gamma interferon (IFN-gamma) in mononuclear cells from PPD skin test-positive individuals. Synergistic suppression of IFN-gamma in cultures containing both rTGF-beta1 and rIL-10 was only seen when the responder cell population were peripheral blood mononuclear cells (PBMC) and not monocyte-depleted mononuclear cells and when PBMC were pretreated with rTGF-beta1 but not with rIL-10. Suppression of PPD-induced IFN-gamma in PBMC containing both rTGF-beta1 (1 ng/ml) and rIL-10 (100 pg/ml) was 1.5-fold higher (P < 0.05) than cultures containing TGF-beta1 alone and 5.7-fold higher (P < 0.004) than cultures containing IL-10 alone. Also, neutralization of endogenous TGF-beta1 and IL-10 together enhanced PPD-induced IFN-gamma in PBMC in a synergistic manner. Thus, TGF-beta1 and IL-10 together potentiate the downmodulatory effect on M. tuberculosis-induced T-cell production of IFN-gamma, and TGF-beta1 alone enhances IL-10 production. At sites of active M. tuberculosis infection, these interactions may be conducive to the suppression of mononuclear cell functions.     

Interaction of immune and connective tissue cells: I. The effect of lymphokines and monokines on fibroblast growth

In order to determine if mononuclear cells may be secreting factors capable of modulating fibroblast growth, the in vitro proliferative response of fibroblasts to cytokines known to be secreted by mononuclear cells was measured, using both growth arrested and proliferating cells. Of the cytokines tested, which included interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), platelet derived growth factor (PDGF), and tumor necrosis factor-alpha (TNF-alpha), only TNF-alpha and PDGF had demonstrable growth factor activity. Neither IL-1 alpha nor beta showed any true growth factor activity but were able to enhance the replication of already proliferating cells. No inhibition of proliferation was noted by any of the cytokines with the exception of TNF-alpha and TGF-beta. TNF-alpha in doses greater than 500 ng/ml caused fibroblast death, probably by a prostaglandin related mechanism as fibroblasts remained viable, although non proliferative, when assayed in the presence of indomethacin, a known inhibitor of prostaglandin E2 (PGE2) synthesis. TGF-beta was inhibitory to proliferation at doses greater than 100 ng/ml, while fibroblasts remained viable. This effect was not influenced by indomethacin and hence is unlikely to be PGE2 related.     

Expression of cytokines and inducible nitric oxide synthase mRNA in the lungs of mice infected with Cryptococcus neoformans: effects of interleukin-12.

We have recently established a murine model of pulmonary and disseminated infection with a highly virulent strain of Cryptococcus neoformans and demonstrated that administration of interleukin-12 (IL-12) protected the animals against infection. In this study, we extended these studies by investigating the host defense mechanisms. In particular, we examined the expression of mRNA for helper T-cell 1 (Th1) cytokines (IL-2, lymphotoxin, and gamma interferon [IFN-gamma]), Th2 cytokines (IL-4, -6, and -10), macrophage-derived cytokines (tumor necrosis factor alpha [TNF-alpha], IL-1beta, transforming growth factor beta [TGF-beta, IL-12p40, and IFN-gamma-inducing factor [IGIF]), and inducible nitric oxide synthase (iNOS) in the lungs on days 1, 3, 7, and 14 after infection and following treatment with IL-12. There was little or no expression of mRNAs for Th1 cytokines, TNF-alpha, IL-12p40, IGIF, and iNOS in the infected mice, but expression increased markedly after treatment with IL-12. In contrast, the mRNAs for Th2 cytokines, IL-1beta, and TGF-beta were detected at considerable levels during the early stages of infection, and, interestingly, expression was not suppressed by IL-12 but rather augmented, particularly during the late stage. Similar results were also obtained for IFN-gamma, IL-4, IL-10, and TNF-alpha measured in the lung homogenates by enzyme-linked immunosorbent assay. These results suggest that the predominance of expression of Th2 cytokines and TGF-beta over Th1 cytokines, TNF-alpha, IL-12p40, IGIF, and iNOS is associated with severe lethal infection in mice and that administration of IL-12 protects infected animals by stimulating Th1 cytokines.     

Role of single nucleotide polymorphisms of cytokine genes in spontaneous preterm delivery

Intra-amniotic infections are implicated in spontaneous preterm delivery (PTD). Certain genetic polymorphisms are associated with increased production of proinflammatory and/or decreased production of anti-inflammatory cytokines, thereby possibly promoting PTD. We determined the relationship between maternal and fetal cytokine gene polymorphisms with occurrence and severity of spontaneous PTD (PTD after spontaneous-onset preterm labor and/or preterm prelabor rupture of membranes) and their association with intrauterine inflammation and infection. DNA from buccal brushings of 80 preterm (gestation < 35 weeks) and 80 matched term mother-infant pairs was assayed for tumor necrosis factor alpha (TNF-alpha [-308G/A]), interferon-gamma (IFN-gamma [+874A/T]), interleukin-6 (IL-6 [-174C/G]), interleukin-10 (IL-10 [-1082G/A, -819C/T, -592C/A]), and transforming growth factor beta1 (TGF-beta1 [T/Ccodon10,G/Ccodon25]) by using polymerase chain reaction (PCR) with sequence-specific primers. The presence of histologic chorioamnionitis was determined for PTDs. Conditioned on maternal IFN-gamma genotypes, fetal high IFN-gamma producing allele (IFN-gamma[+874T]) was associated with spontaneous PTD (odds ratio = 2.3 [1.2-4.4]). Among preterm deliveries, maternal low TGF-beta1 (TGF-beta1 [codon10C]) producing genotypes correlated negatively with gestation. Fetal TNF-alpha (-308G) was significantly associated with histologic chorioamnionitis. Underlying genitourinary infections and/or inflammation were significantly associated with maternal and fetal IL-6 (-174G), fetal TNF-alpha (-308GG), and fetal IL-10 (-1082A). We conclude that certain fetal and maternal cytokine gene polymorphisms may be associated with occurrence and/or severity of spontaneous PTD and with intrauterine inflammation and infection.     

Coordinate cytokine gene expression in vivo following induction of tuberculous pleurisy in guinea pigs

Tuberculous pleurisy is a severe inflammatory response induced by Mycobacterium tuberculosis organisms that have escaped from lung granulomata into the pleural space during pulmonary infection. We have used the guinea pig model of tuberculous pleurisy to examine several aspects of the immune response to this antigen-specific inflammatory event. Pleurisy was induced by injection of heat-killed M. tuberculosis H37Rv directly into the pleural space of guinea pigs previously vaccinated with M. bovis BCG. Four animals were euthanized each day over a period of 9 days. Fluid in the pleural cavity was analyzed for transforming growth factor beta 1 (TGF-beta 1) and total interferon (IFN) protein levels. In addition, RNA was obtained from pleural cells and examined for TGF-beta 1, tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and interleukin-8 (IL-8) expression by real-time PCR. Finally, pleural cells were examined for the ability to proliferate in response to concanavalin A and purified protein derivative (PPD) in vitro. In the pleural fluid, TGF-beta 1 protein concentrations increased over the course of the inflammatory response while IFN protein levels were not significantly altered. Expression of TGF-beta 1 mRNA peaked on days 3 and 4, and IFN-gamma mRNA expression peaked on day 3 and then returned to background levels. TNF-alpha mRNA expression was highest on days 2 to 4, and IL-8 mRNA levels remained elevated between days 2 and 5, peaking on day 3 before returning to background levels. PPD-induced proliferative responses were evident by day 3 and remained present throughout the study. Analysis of cytokine expression during tuberculous pleurisy may lead to a better understanding of the self-healing nature of this manifestation of tuberculosis.     

Cytokine-associated tissue injury and lethality in mice: a comparative study

A comparative study was performed to examine the lethal effects of several cytokines injected into mice sensitized with actinomycin D (Act-D). Consistent with published data, human tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) (0.2-5 micrograms) caused the death of the animals within 8-12 hr after injection. Human interleukin-6 (IL-6) and interleukin-8 (IL-8) (0.6-6 micrograms) known to be induced by TNF-alpha did not show any lethal effects, indicating that TNF-alpha-associated lethality is not mediated by IL-6 or IL-8. Human tumor necrosis factor-beta (TNF-beta) (also called lymphotoxin), which shares structural and functional properties with TNF-alpha, was as potent as TNF-alpha in its lethal effects. Murine interferon-gamma (IFN-gamma) (0.04-5 micrograms) was also tested and showed no lethal effects in this model. In addition, a synthetic peptide corresponding to amino acid residues 163-171 of IL-1 beta, and which has been shown to lack the inflammatory effects of IL-1 beta, also caused no lethality among Act-D sensitized mice. The pretreatment of mice with IL-6, IL-8, or IFN-gamma had no protective effects on TNF-alpha or IL-1 beta-induced lethality in contrast to the protection observed by a pretreatment with TNF-alpha/IL-1 beta themselves or with endotoxin. Histopathologic data showed that severe tissue injury in vital organs is associated with the rapid lethality among sensitized mice.     

Cytokine regulation of intercellular adhesion molecule-1 (ICAM-1) expression on human glioblastoma cells

Intercellular adhesion molecule-1 (ICAM-1) has recently been identified as one of the ligands for lymphocyte function-associated antigen-1 (LFA-1). Immunohistochemical staining of frozen tissue sections using the ICAM-1 antibody RR1/1 demonstrated significant levels of ICAM-1 expression on human glioblastoma cells and on intratumoural vascular endothelial cells. ICAM-1 was weakly expressed or absent from low grade gliomas and absent from normal and fetal brain. ICAM-1 expression was similar to that of MHC class II. HLA-DR antigens. Glioblastoma cell lines constitutively expressed ICAM-1 to a minimal or moderate extent. Surface antigen expression of ICAM-1 and ICAM-1-specific mRNA could be significantly increased by incubating glioblastoma cells with interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma). IL-2, IL-4, IL-6 and transforming growth factor beta 2 (TGF-beta 2) had no significant effect on surface antigen expression. Significant enhancement of ICAM-1 expression was obtained using TNF-alpha and IL-1 beta at 1-10 U/ml and at 500 U/ml of IFN-gamma. Induction of ICAM-1 specific mRNA was observed 4 h after cytokine treatment and decreased by 24 h. Surface antigen expression of ICAM-1 increased for up to 48 h after treatment.     

Suppression of lymphokine-activated killer (LAK) cell induction mediated by interleukin-4 and transforming growth factor-beta 1: effect of addition of exogenous tumour necrosis factor-alpha and interferon-gamma, and measurement of their endogenous production.

Recombinant human interleukin-4 (rhIL-4) and transforming growth factor-beta 1 (TGF-beta 1) suppressed the induction of lymphokine-activated killer (LAK) activity induced by recombinant human interleukin-2 (rhIL-2) in peripheral blood lymphocytes. DNA synthesis and the expression of the p55 alpha chain of the IL-2 receptor (Tac antigen) were also inhibited. The inhibitory effect was greatest when these factors were added during the first 48 h of a 4-day culture, with reduced cytolytic activity against both natural killer (NK) resistant and NK-sensitive tumour cell line targets. The suppressive action of both cytokines was accompanied by a reduction in tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) levels in lymphocyte culture supernatants. Recombinant human IFN-gamma (rhIFN-gamma), but not recombinant human TNF-alpha (rhTNF-alpha) was able to overcome the inhibitory effect of recombinant human interleukin-4 (rhIL-4) on LAK induction and DNA synthesis but not Tac antigen expression. However, cytotoxicity induced by rhIFN-gamma alone was also suppressed by rhIL-4 and TGF-beta 1, inferring that rhIFN-gamma-mediated abrogation of rhIL4 suppression was not simply a direct IL-2-independent effect on cytotoxicity. In addition, rhIL-4 did not increase TGF-beta production from rhIL-2-activated peripheral blood mononuclear cells, suggesting that rhIL-4 did not mediate reduction of rhIL-2 responses through the induction of TGF-beta release.     

Modulation of human microglia and THP-1 cell toxicity by cytokines endogenous to the nervous system

Neuroinflammatory processes are thought to be a significant factor in the pathology of a number of degenerative neurological diseases. A variety of cytokines influence inflammatory levels. Here we show that a cooperative action of two or more cytokines is required to induce significantly human microglial and monocytic THP-1 cell toxicity towards SH-SY5Y neuroblastoma cells. Such toxicity was induced by the following combinations: interferon-gamma (IFN-gamma) with tumor necrosis factor-alpha (TNF-alpha); IFN-gamma with interleukin (IL) 1alpha or IL-1beta in the presence of TNF-alpha; and IL-6 with TNF-alpha. Toxicity induced by the various stimulatory combinations was not accompanied by an increased nitrite production. Of the potential inhibitors tested, IL-4 downregulated the toxic action of microglia when applied to THP-1 cells either before stimulation or 24 h after stimulation. Toxicity was not inhibited by IL-10, and was even enhanced by transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF). These data suggest that antagonists of cytokine receptors, as well as inhibitors of their intracellular pathways may be effective anti-inflammatory agents.     

Activation of human natural killer cells by lipopolysaccharide and generation of interleukin-1 alpha, beta, tumour necrosis factor and interleukin-6. Effect of IL-1 receptor antagonist.

The presence in the body of an antigen species or a bacterial lipopolysaccharide (LPS) has a pleiotropic effect on the immune system activating macrophages, lymphocytes and natural killer (NK) cells. Recently it has been reported that human macrophages not only secrete interleukin-1 (IL-1) but also its inhibitor, called IL-1 receptor antagonist (IL-1ra), structurally similar to IL-1 beta, but with no IL-1-like activity and which binds to the IL-1 receptor. In this study we show that LPS stimulates NK cell activity and IL-1ra potentiates the stimulatory effect of human recombinant interleukin-2 (hrIL-2) on NK cell activity. In addition, we found that hrIL-1ra inhibits DNA synthesis in lymphocyte culture stimulated with phytohaemagglutinin (PHA) (20 micrograms/ml), presumably via IL-1 inhibition. We also found that LPS is a potent stimulator of monokines: IL-6, tumour necrosis factor-alpha (TNF-alpha), and IL-1 beta, as determined by radioimmunoassay method, and interferon-gamma (IFN-gamma), IL-2, TNF-alpha and IL-1 alpha, as determined by ELISA method, in peripheral blood mononuclear cells (PBMC). We used PBMC as effector cells since LPS requires the presence of accessory cells to activate lymphocytes and bind to the HLA-DR molecule on accessory cells. The effect of LPS on PBMC cytotoxicity has been compared with an endotoxin-free extract of Escherichia coli, OM-8990, which did not provoke cytokine production nor did it cause enhancement of NK cell activity. We found that human recombinant IL-1ra potentiates the stimulatory effect of IL-2 on NK cell activity, similar to hrIL-1 beta. The potentiation of IL-2 in stimulating NK cell activity by IL-1ra is not yet understood. Since IL-1ra is a part of the IL-1 family, it may work in a similar fashion to IL-1, which also potentiates IL-2 to enhance NK cell activity but has been shown not to be directly important in tumour cell killing. In addition, hrIL-1ra can amplify the effect of IL-2 on NK activity, possibly by inhibiting the cyclo-oxygenase products, which are immunosuppressive and are generated in antigen-stimulated PBMC cultures. The generation of IFN-gamma by PBMC after treatment with LPS strongly suggests that the enhancement of NK cell activity may be indirectly due to IFN production.     

Evidence for cytokine dysregulation in multiple sclerosis: peripheral blood mononuclear cell production of pro-inflammatory and anti-inflammatory cytokines during relapse and remission

We investigated circulating anti-inflammatory and pro-inflammatory cytokines, and their ex vivo PBMC production in the absence or presence of the neuroantigens myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) and T cell mitogen (PHA) in MS patients in relapse and remission, patients with other neurological disorders (OND) and normal healthy controls. MS patients in relapse exhibited significantly increased PBMC production of TNF-alpha spontaneously compared with MS remission and healthy controls and with MBP compared with MS remission. Patients in relapse had significantly increased spontaneous, PHA- and MBP-induced PBMC IL-1beta production compared with remission MS, and was increased compared (PHA only) with OND and healthy controls. In relapse there was also significantly increased PBMC IFN-gamma production (PHA only) compared with remission and a significantly lower production of biologically active TGF-beta1 (PHA only) compared with remission MS and OND. In contrast, MS patients in remission produced significantly less spontaneous and MBP-induced TNF-alpha, spontaneous, PHA- and MBP-induced IL-1beta and PHA-induced IFN-gamma together with increased production of biologically active TGF-beta1. MOG non-specifically increased PBMC TNF-alpha and IL-1beta production in all groups. Pro-inflammatory cytokines in corresponding plasma samples were undetectable whilst the concentration of biologically active TGF-beta1 was the reverse of ex vivo PBMC findings. The increase in biologically active TGF-beta1 production ex vivo in OND patients, despite active disease, compared with the low level in the MS relapse may indicate a regulatory defect in MS. We conclude that the balance between biologically active TGF-beta1 and the pro-inflammatory TNF-alpha, IL-1beta and IFN-gamma is dysregulated during MS relapse-remission and that normal counter-regulatory mechanisms during the relapse phase are defective.     

TH2 Cytokine-enhanced and TGF-beta-enhanced vascular endothelial growth factor production by cultured human airway smooth muscle cells is attenuated by IFN-gamma and corticosteroids

BACKGROUND: T(H)2 and T(H)1 cytokines have opposite effects on many aspects of the inflammatory response. METHODS: This study was designed to determine if cytokines possibly present in asthma can modulate airway smooth muscle cell (ASMC) production of vascular endothelial growth factor (VEGF) and thus contribute to altered airway vascularity. ASMC were incubated for 24 hours with various concentrations of T(H)2 cytokines (IL-4, IL-5, IL-10, and IL-13); transforming growth factor (TGF)-beta1, TGF-beta2, or TGF-beta3; and IL-1beta or TNF-alpha with or without IFN-gamma. Budesonide and exogenous prostaglandin (PG)E(2) were also evaluated. Postculture media were assayed for VEGF and PGE(2) by ELISA. RESULTS: IL-4, IL-5, and IL-13 alone but not IL-10 enhanced VEGF production by ASMC in a concentration-dependent manner. IFN-gamma alone inhibited spontaneous VEGF release by ASMC and concentration-dependently attenuated IL-4-augmented, IL-5-augmented, or IL-13-augmented production of VEGF (P <.01). All three TGF-beta isoforms augmented VEGF production, which was reduced by IFN-gamma (P <.005). IL-1beta also increased VEGF production, but this was not affected by IFN-gamma (P >.05). TNF-alpha alone had little effect on VEGF release by ASMC. Production of VEGF stimulated by all cytokines was inhibited by budesonide. Exogenous PGE(2) increased VEGF release, but cytokine modulation of PGE(2) release did not always correlate with VEGF release. CONCLUSIONS: T(H)2 cytokines and TGF-beta stimulate ASMC release of VEGF. This can be inhibited by IFN-gamma and glucocorticoids.     

Pathology of Plasmodium chabaudi chabaudi infection and mortality in interleukin-10-deficient mice are ameliorated by anti-tumor necrosis factor alpha and exacerbated by anti-transforming growth factor beta antibodies

Interleukin-10 (IL-10)-deficient (IL-10(-/-)) mice infected with Plasmodium chabaudi (AS) suffer a more severe disease and exhibit a higher rate of mortality than control C57BL/6 mice. Here, we show that a drop in body temperature to below 28 degrees C and pronounced hypoglycemia of below 3 mM are reliable indicators of a lethal infection. Elevated inflammatory responses have been shown to accompany pathology in infected IL-10(-/-) mice. We show that neutralization of tumor necrosis factor alpha (TNF-alpha) in IL-10(-/-) mice abolishes mortality and ameliorates the hypothermia, weight loss, and anemia but does not affect the degree of hypoglycemia. These data suggest that TNF-alpha is involved in some of the pathology associated with a P. chabaudi infection in IL-10(-/-) mice but other factors play a role. IL-10(-/-) mice that survive a primary infection have been shown to control gamma interferon (IFN-gamma) and TNF-alpha production, indicating that other cytokines or mechanisms may be involved in their down-regulation. Significantly higher levels of transforming growth factor beta (TGF-beta), a cytokine with such properties, are present in the plasma of infected IL-10(-/-) mice at a time that coincides with the disappearance of IFN-gamma and TNF-alpha from the blood. Neutralization of TGF-beta in IL-10(-/-) mice resulted in higher circulating amounts of TNF-alpha and IFN-gamma, and all treated IL-10(-/-) mice died within 12 days with increased pathology but with no obvious increase in parasitemia. Our data suggest that a tight regulation of the balance between regulatory cytokines such as IL-10 and TGF-beta and inflammatory cytokines such as IFN-gamma and TNF-alpha is critical for survival in a mouse malaria infection.     

Effects of 2-deoxy-D-glucose administration on cytokine production in BDF1 mice.

Physical exercise and diet changes have been shown to affect immune parameters, and similar effects are also induced by the administration of a nonmetabolizable glucose analog, 2-deoxy-D-glucose (2-DG). The present study was designed to characterize the effects of glucoprivation induced by 2-DG administration on concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in the blood and interferon-gamma (IFN-gamma), IL-2, and IL-4 in vitro production by partially purified T splenocytes in BDF1 mice. Mice (n = 8 per group) were injected intraperitoneally one or three times with 0, 500, 750, or 1000 mg/kg of 2-DG, and blood and spleens were collected 2 h after the last injection. Partially purified T splenocytes were cultured 24 h in the presence of concanavalin A (ConA). A significant increase in the corticosterone levels with the amount of 2-DG injected was observed after one or three injections (p<0.05). The amount of 2-DG injected was associated with an increase in TNF-alpha, IL-1beta, and IL-6 concentrations in the blood of mice after one or three injections of 2-DG (p<0.05). A significant decrease in in vitro proliferation of partially purified splenocytes in the presence of ConA was associated with a decrease in IFN-gamma production in the culture supernatants and an increase in IL-1 receptor expression on the cell surface (p<0.05).     

Cytokine sensitivity of Langerhans' islets of diabetes-prone BB/OK rats under hypoglycemic conditions

Islets of Langerhans isolated from diabetes-prone BB/OK rats were exposed to interleukin-1beta (IL-1beta) or to a combination of tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) under hypoglycemia at glucose concentrations of 2.2 and 3.2 mmol/l or in the presence of stimulatory conditions at 6.0 and 11 mmol/l glucose. For estimating cytokine effects the islets were functionally assayed by measurement of glucose stimulated insulin secretion. Pancreatic islets exposed for 24 h to IL-1beta at a glucose concentration of 6.0 mmol/l exhibited a reduced insulin secretion following a 48h recovery period compared to islets which were cytokine treated at 2.2 or 3.2mmol/l glucose, respectively. Islets pre-exposed for 24h to TNF-alpha plus IFN-gamma at 2.2, 3.2 or 6.0 mmol/l glucose displayed no alterations of insulin secretion following a 48 h regeneration. A temporary (3 h) influence of IL-1beta under hyperglycemic conditions at 11 mmol/l glucose caused a reduction of the subsequent insulin secretion of Langerhans' islets prior incubated for 24 h at 6.0 mmol/l glucose without cytokines, but not of islets precultured at 2.2 mmol/l glucose. In contrast, a 3 h treatment with TNF-alpha plus IFN-gamma at 11 mmol/l glucose did not affect insulin secretion of islets prior held at 6.0 mmol/l glucose, whereas a transient exposure for 6h to IL-1beta as well as TNF-alpha plus IFN-gamma under similar conditions diminished insulin secretion of islets preincubated at 2.2 or 6.0 mmol/l glucose. In conclusion, hypoglycemia reduces the sensitivity of BB/OK rat islets to IL-1beta, whereas a slight elevation of glucose concentration to 6.0 mmol/l increases again their vulnerability. TNF-alpha plus IFN-gamma at concentrations capable to decrease insulin secretion of islets during hyperglycemia do not affect the insulin output in a range between 2.2 and 6.0 mmol/l glucose. During glucose stimulation at 11 mmol/l islets' insulin secretory machinery is protected from IL-1beta as well as TNF-alpha plus IFN-gamma for 3 h by a preceding 24 h hypoglycemia, but its vulnerability is restored within additional 3 h.