The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.     

Analysis of cytokine mRNA profiles in the lungs of Pneumocystis carinii-infected mice

Severe combined immunodeficient (scid) mice lack functional CD4+ lymphocytes, and therefore develop life-threatening Pneumocystis carinii infection. However, when scid mice are immunologically reconstituted with spleen cells, including CD4+ cells, a protective inflammatory response is mounted against the organism. To determine whether these lymphocytes induce elevated cytokine mRNA levels in response to P. carinii infection, steady-state levels of cytokine mRNAs were measured in the lungs of both reconstituted and unaltered scid mice. Despite significant numbers of organisms and the presence of functional alveolar macrophages in the lungs of 8- and 10-wk-old scid mice, there was neither evidence of pulmonary inflammation, nor increased proinflammatory cytokine expression. However, when 8-wk-old scid mice were immunologically reconstituted, signs of intense, focal pulmonary inflammation were observed, and levels of interleukin (IL)-1alpha, IL-1beta, IL-3, IL-6, interferon-gamma (IFN-gamma), tumor necrosis factor (TNF)-alpha, and TNF-beta mRNAs were all significantly elevated. Cytokine expression was increased at day 10 post-reconstitution (PR), maximal at day 12 PR, and returned to baseline by day 22 PR. In situ hybridization demonstrated that at day 12 PR, increased IL-1beta and TNF-alpha expression was localized to sites of intense inflammation and focal P. carinii colonization. Many of the cells expressing high levels of IL-1beta and TNF-alpha in these regions were in direct contact with organisms, or contained degraded organisms within their cytoplasm. Thus, even though functional macrophages are present in scid mice, CD4+ T cells are required for proinflammatory cytokine expression, which is associated with the generation of a protective inflammatory response at sites of P. carinii infection.     

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.     

Topical FK506 suppresses cytokine and costimulatory molecule expression in epidermal and local draining lymph node cells during primary skin immune responses

Recently, it has been shown that the immunosuppressive macrolide lactone, FK506, exerts good therapeutic efficacy in inflammatory skin diseases. The aim of this study was to analyze the influence of topical FK506 on molecular (IL-1alpha, IL-1beta, IL-2, IL-4, IL-12 p35, IL-12 p40, macrophage inflammatory protein-2 (MIP-2), granulocyte-macrophage CSF (GM-CSF), TNF-alpha, and IFN-gamma) and cellular (I-A+/CD80+, I-A+/CD54+, I-A+/CD69+, I-A+/B220+, and CD4+/CD25+) events in epidermal (EC) and local draining lymph node (LNC) cells during primary contact hypersensitivity responses. Cytokine mRNA levels for IL-1alpha, IL-1beta, GM-CSF, TNF-alpha, MIP-2, and IFN-gamma in EC and for IL-2, IL-4, IL-12 p35, IL-12 p40, and IFN-gamma in LNC were increased and resulted in significant LNC proliferation during oxazolone-induced contact hypersensitivity. Topical FK506 treatment dose-dependently suppressed oxazolone-induced LNC proliferation. This effect was correlated with decreased IL-1alpha, IL-1beta, GM-CSF, TNF-alpha, MIP-2, and IFN-gamma mRNA expression within the epidermis and decreased IL-12 p35 and p40 mRNA expression in LNC. Further analysis of the LNC cytokine pattern revealed that the production of both Thl (IFN-gamma and IL-2) and Th2 (IL-4) cytokines was dramatically impaired after topical FK506 treatment. Flow cytometric analysis showed that topical FK506 decreased the population of epidermis-infiltrating CD4+ T cells and suppressed the expression of CD54 and CD80 on I-A+ EC and LNC during hapten-induced contact hypersensitivity. Furthermore, topical FK506 profoundly impaired oxazolone-induced up-regulation of CD25 expression on CD4+ LNC and dramatically decreased hapten-induced expansion of I-A+/B220+ and I-A+/CD69+ LNC subsets. In conclusion, these results give new insights into the mechanisms of action of topical FK506 treatment.     

Th1 response in Salmonella typhimurium-infected mice with a high or low rate of bacterial clearance

Previous studies have shown that the capacity to clear an attenuated strain of Salmonella typhimurium after the second week of infection varies widely among mouse strains. Bacterial clearance is mediated by CD4+ T cells and is regulated in part by the H-2 complex. The aim of the present study was to compare the patterns of cytokine mRNA expression in the spleens of C57BL/6 (H-2b) and CBA (H-2k) mice, which exhibit a low and a high rate of bacterial clearance, respectively. A transient increase in interleukin-12 (IL-12) mRNA levels was found in both mouse strains. Gamma interferon (IFN-gamma) gene expression was higher and more sustained in C57BL/6 than in CBA mice. No increase in IL-4 mRNA was detected. A transient increase in IL-10 mRNA was found in C57BL/6 mice. Separation of spleen cells into CD4+ and CD4- fractions showed that CD4+ T cells produced the bulk of IFN-gamma in both mouse strains and of IL-10 in C57BL/6 mice. Infection of H-2 congenic mice induced a higher level of IFN-gamma mRNA expression by CD4+ T cells in mice with a low rate of clearance (H-2b) than in mice with a high rate of clearance (H-2q). Treatment of infected C57BL/6 mice with anti-IFN-gamma or anti-CD4 monoclonal antibodies indicated that IFN-gamma participates in resistance in the early phase of infection, but not in bacterial clearance, and that CD4+ T cells mediate bacterial clearance during the 3rd week of infection. Taken together, these results suggest that defective bacterial clearance in H-2b mice is not linked to defective IFN-gamma production and that CD4+ T cells mediate bacterial clearance by an IFN-gamma-independent mechanism.     

The mRNA-phenotype of granuloma formation: CD4+ T cell-associated cytokine gene expression during primary murine listeriosis

In murine listeriosis, elimination of bacteria and immunity to reinfection critically depend on Thy1+ CD4- cells, while cell-mediated inflammatory phenomena such as DTH and granuloma formation are mostly mediated by CD4+ T cells. In an attempt to correlate T cell phenotype and function with a particular set of cytokines produced, we examined the cytokine gene expression profile associated with the presence or absence of Thy1+, CD4+ and/or CD8+ cells in the livers of mice during a primary infection with L. monocytogenes. The presence of CD4+ cells was found to be closely associated with mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increase in expression of TNF-alpha and GM-CSF and a 25-fold increase in expression of IFN-gamma and TNF-beta mRNAs, and temporally coincided with the development of granulomatous lesions. In vivo neutralization of TNF-alpha and, to a lesser extent, IFN-gamma resulted in abrogation of granuloma formation. A similar correlation between the presence of CD8+ cells and mRNA expression for any one of the cytokines studied did not exist, pointing to a qualitatively different mechanism of CD8+ T cell mediated cure of listeriosis.     

TNF-alpha down-regulates type 1 cytokines and prolongs survival of syngeneic islet grafts in nonobese diabetic mice

Administration of TNF-alpha to autoimmune diabetes-prone nonobese diabetic mice and biobreeding rats inhibits diabetes development; however, the mechanism(s) of diabetes prevention by TNF-alpha has not been established. We used the model of syngeneic islet transplantation into diabetic nonobese diabetic mice to study the effects of TNF-alpha administration on the types of mononuclear cells and cytokines expressed in the islet grafts and on autoimmune diabetes recurrence. Twice daily i.p. injections of TNF-alpha (20 microg/day) from day 1 to day 30 after islet transplantation significantly prolonged islet graft survival; thus, 70% (16 of 23) of mice treated with TNF-alpha were normoglycemic at 30 days after islet transplantation compared with none (0 of 14) of vehicle-treated control mice. Islet grafts and spleens from TNF-alpha-treated mice at 10 days after islet transplantation contained significantly fewer CD4+ and CD8+ T cells, and significantly decreased mRNA levels of type 1 cytokines (IFN-gamma, IL-2, and TNF-beta) than islet grafts and spleens from control mice. Regarding type 2 cytokines, IL-4 mRNA levels were not changed significantly in islet grafts or spleens of TNF-alpha-treated mice, whereas IL-10 mRNA levels were decreased significantly in islet grafts of TNF-alpha-treated mice and not significantly changed in spleens. TGF-beta mRNA levels in islet grafts and spleens were similar in TNF-alpha-treated and control mice. These results suggest that TNF-alpha partially protects beta cells in syngeneic islet grafts from recurrent autoimmune destruction by reducing CD4+ and CD8+ T cells and down-regulating type 1 cytokines, both systemically and locally in the islet graft.     

Differential localization of Th1 and Th2 cells in autoimmune gastritis

The vast majority of CD4+ T cells infiltrating into gastric mucosa (GM) and in the draining (gastric) lymph node (GLN) shows an activated/memory phenotype, CD45RB(low) L-selectin(low) CD44(high), in neonataly thymectomized BALB/c mice bearing autoimmune gastritis (AIG), indicating that these cells are actively involved in this disease. CD4+ T cells sort-purified from GLN expressed mRNAs encoding for both IFN-gamma and IL-4. However, those infiltrating into GM expressed very low levels of IL-4 mRNA, even though they strongly expressed IFN-gamma mRNA. Among CD4+ T cells separated from AIG mice expressing detectable levels of either IFN-gamma or IL-4 by intracellular staining, less than one-seventh expressed IL-4 and thus most of them expressed IFN-gamma in GM, whereas roughly half and one-third expressed IL-4 in GLN and spleen respectively. These findings indicate that the Th1 cells predominantly infiltrate into autoimmune lesions and Th2 cells are mainly resident in the regional LN. We further set up an in vitro model system of transendothelial migration using a murine endothelial cell line, F-2, and found that Th1 cells in CD4+ T cells separated from lymphoid tissues of AIG mice preferentially passed through the monolayer of endothelial cells while only a small portion of Th2 cells did so. This differing ability of transendothelial migration and localization might explain the dominance of Th1 cells destroying the tissue in focal lesions without inhibition by the Th2 cells, in spite of both subsets being simultaneously activated in AIG mice, and the functions of each T cell subset seems to be mutually exclusive.     

Association of CD4+ T cell-dependent keratitis with genetic susceptibility to Pseudomonas aeruginosa ocular infection

The role of T lymphocytes in susceptibility to Pseudomonas aeruginosa corneal infection was studied in inbred C57Bl/6 (B6) beta2-microglobulin+/+ (beta2m+/+) and beta2m-/- knockout (KO) mice on a B6 genetic background. The corneas of both B6 and KO mice perforated by 7 days postinfection (p.i.). Histopathology revealed a similar inflammatory response characterized by an infiltration of polymorphonuclear neutrophilic leukocytes by 24 h p.i. in both groups of mice. CD4+ and CD8+ (latter absent in KO) T cells were present in cornea by 3 days p.i., and by 5 days, IL-2R-positive cells were positively immunostained. Corneas of B6 beta2m+/+ mice depleted of CD4+ T cells and infected with P. aeruginosa did not perforate at 7 days p.i. vs mice depleted of CD8+ T cells or treated with an irrelevant mAb. Neutralization of IFN-gamma before infecting B6 mice prevented corneal perforation and was associated with a lower delayed-type hypersensitivity than in B6 mice similarly treated with an irrelevant mAb. These data provide evidence that a CD4+ T cell (Th1)-dominated response following P. aeruginosa corneal infection is associated with genetic susceptibility and corneal perforation in inbred B6 mice.     

Increased in vitro induced CD4+ and CD8+ T cell IFN-gamma and CD4+ T cell IL-10 production in stable relapsing multiple sclerosis

Multiple sclerosis (MS) is presumed to be a T-cell mediated chronic inflammatory disease of the central nervous system. Investigators previously demonstrated increased IFN-gamma (pro-inflammatory) and IL-10 (counterregulatory anti-inflammatory) in MS. The balance of pro-inflammatory and counterregulatory anti-inflammatory cytokines may be important in the stabilization of disease activity. Purified CD4+ and CD8+ T cells from patients with clinically definite, stable relapsing MS (RRMS) were stimulated by anti-CD3 mAb or Con A for 48 hours and cytokine supernatants analysed for production of IL-2, IL-6, IFN-gamma, TNF-alpha (potential pro-inflammatory) and IL-4, IL-10, and TGF-beta (potential counterregulatory anti-inflammatory). Con A activated CD4+ and CD8+ T cell proinflammatory cytokine IL-2 secretion, CD4+ T cell IL-6 secretion, CD4+ and CD8+ T cell TNF-alpha secretion and CD8+ T cell IFN-gamma secretion was decreased significantly in RRMS subjects compared to controls. CD3 activated CD4+ and CD8+ T cell IL-6 secretion and CD4+ T cell TNF-alpha secretion was significantly decreased in MS subjects compared to controls. In contrast, there was increased CD3-induced IFN-gamma in both CD4+ and CD8+ T cells and counterregulatory anti-inflammatory CD3-induced IL-10 secretion in CD4+ T cells in RRMS compared to controls. These data suggest that an equilibrium of a pro-inflammatory (IFN-gamma) and a counterregulatory anti-inflammatory (IL-10) cytokine may define stable clinically definite early RRMS.     

The inflammatory response to nonfatal Sindbis virus infection of the nervous system is more severe in SJL than in BALB/c mice and is associated with low levels of IL-4 mRNA and high levels of IL-10-producing CD4+ T cells

SJL mice are susceptible to inflammatory autoimmune diseases of the central nervous system (CNS), while BALB/c mice are relatively resistant. To understand differences in immune responses that may contribute to autoimmune neurologic disease, we compared the responses of SJL and BALB/c mice to infection with Sindbis virus, a virus that causes acute nonfatal encephalomyelitis in both strains of mice. Clearance of virus was similar, but SJL mice developed a more intense inflammatory response in the brain and spinal cord and inflammation persisted for several weeks. Analysis of lymphocytes isolated from brains early after infection showed an absence of NK cells in SJL mice, while both strains of mice showed CD4+ and CD8+ T cells. During the second week after infection, CD4+ T cells increased in SJL mice and the proportion of CD8+ T cells decreased, while the opposite pattern was seen in BALB/c mice. Expression of IL-10 mRNA was higher and IL-4 mRNA was lower in the brains of infected SJL than in BALB/c mice, while expression of the mRNAs of IL-6, IL-1beta, TNFalpha, and the Th1 cytokines IL-2, IL-12, and IFN-gamma was similar. Lymphocytes isolated from the CNS of SJL mice produced large amounts of IL-10. CNS lymphocytes from both strains of mice produced IFN-gamma in response to stimulation with Sindbis virus, but not in response to myelin basic protein. These data suggest that IL-10-producing CD4+ T cells are differentially recruited to or regulated within the CNS of SJL mice compared with BALB/c mice infected with Sindbis virus, a characteristic that may be related to low levels of IL-4, and is likely to be involved in susceptibility of SJL mice to CNS inflammatory diseases.     

Mice lacking the IFN-gamma receptor have impaired ability to resolve a lung eosinophilic inflammatory response associated with a prolonged capacity of T cells to exhibit a Th2 cytokine profile

To investigate the modulatory role of IFN-gamma on the induction and maintenance of Th2 mucosal immunity in vivo, experiments were performed in mice lacking the IFN-gamma R. Aerosol OVA challenge of immunized wild-type mice resulted in an infiltration of eosinophils into the lung, associated with the ex vivo production of Th2 cytokines (IL-4 and IL-5) from purified lung Thy1.2+ cells stimulated via the CD3/TCR complex. However, while immunized IFN-gamma R-deficient mice exhibited elevated levels of IgE, IgG1, and reduced levels of IgG2a compared with wild-type mice, there was no difference in the recruitment of eosinophils into the lung or the production of IL-4 and IL-5 from lung T cells on day 3. In contrast, up to 2 mo after a single Ag challenge, eosinophils were still present in the lungs of IFN-gamma R-deficient, but not wild-type, mice. Likewise, lung-derived T cells from IFN-gamma R-deficient mice produced higher levels of IL-4 and IL-5, both at 1 and 2 mo after OVA challenge compared with T cells from wild-type mice. We conclude that endogenous IFN-gamma regulates the humoral isotype Ab pattern, but does not modulate the commitment of T cells to a Th2 phenotype in vivo or the acute infiltration of eosinophils to the lung. However, in the absence of IFN-gamma-mediated signaling, there is a transition from a spontaneously resolving to a persisting eosinophilic inflammation of the lungs, associated with a sustained capacity of lung T cells to secrete a Th2 cytokine profile.     

IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFN-gamma and TNF-alpha

IL-17 is a novel T cell-derived cytokine that can regulate the functions of a variety of cell types. In this study, we investigated whether hapten-specific T cells isolated from patients with allergic contact dermatitis (ACD) to nickel produce IL-17 and the effects of IL-17 alone or in combination with IFN-gamma or TNF-alpha on the immune activation of keratinocytes. Skin affected with ACD to nickel and skin-derived, nickel-specific CD4+ T cell lines expressed IFN-gamma, TNF-alpha, and IL-17 mRNAs. Four of seven nickel-specific CD4+ T cell clones positive for the skin-homing receptor, cutaneous lymphocyte-associated Ag, were shown to corelease IL-17, IFN-gamma, and TNF-alpha. In contrast, two nickel-specific CD8+ T cell clones failed to synthesize IL-17. Normal human keratinocytes were found to express constitutively the IL-17 receptor gene. IL-17 specifically and dose-dependently augmented IFN-gamma-induced ICAM-1 expression on keratinocytes at both the mRNA and the protein level, whereas HLA-DR, MHC class I, and CD40 levels were not modulated by IL-17. On the other hand, IL-17 alone did not affect ICAM-1 or enhance TNF-alpha-induced ICAM-1. In addition, IL-17, both directly and in synergism with IFN-gamma and/or TNF-alpha, stimulated synthesis and release of IL-8 by keratinocytes. In contrast, IFN-gamma- and TNF-alpha-induced production of RANTES was markedly inhibited by IL-17, and the synthesis of macrophage chemotactic protein 1 was not changed. Taken together, the results suggest that IL-17 is an important player of T cell-mediated skin immune responses, with synergistic or antagonist effects on IFN-gamma- and TNF-alpha-stimulated keratinocyte activation.     

IL-6, IFN-gamma and TNF-alpha production by liver-associated T cells and acute liver injury in rats administered concanavalin A

The relationship between the development of acute hepatitis and the production of TNF-alpha IFN-gamma and IL-6 by liver-associated T lymphocytes following intravenous injection of concanavalin A (Con A) was studied in rats. Following a single injection of Con A, there was a dose and time-dependent correlation in the serum levels of serum alanine aminotransferase (ALT), IL-6, IFN-gamma and TNF-alpha. These increases correlated with an increase in the numbers of CD4+, CD8+ and CD25+ T cells in blood and CD4+ and CD25+ T cells in the liver perfusate, but not with CD8+ T cells in liver perfusate. Increased levels of IL-6, IFN-gamma and TNF-alpha were constitutively produced by liver-associated CD4+ T cells when cultured. In Con A-stimulated cultures, liver-associated CD4+ T cells secreted increasing levels of TNF-alpha in a time-dependent manner following Con A injection, but TNF-alpha production by peripheral blood lymphocytes was transient with peak levels detected at 1 h which then declined over 24 h. Histological examination of the liver revealed fatty change, hepatocyte degeneration and necrosis, with an associated cell infiltrate of neutrophils and CD4+ T cells both in the portal areas and around the central veins. These results support the hypothesis that Con A-induced liver damage is mediated by CD4+ T cells acting within the liver, at least in part through the secretion of TNF-alpha, IFN-gamma and IL-6.     

Decreased drug accumulation in a mitoxantrone-resistant gastric carcinoma cell line in the absence of P-glycoprotein

The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) beta 2 subunit expression. To determine the basis for this selective loss, we examined IL-12R beta 2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R beta 2 is not expressed by naive resting CD4+ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation. IL-4 inhibited IL-12R beta 2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production. Thus, IFN-gamma may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.     

Human retinal pigment epithelial cell interleukin-8 and monocyte chemotactic protein-1 modulation by T-lymphocyte products

PURPOSE: The purpose of the study was to examine the effect of T-lymphocyte products on human retinal pigment epithelial (HRPE) cell interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and gene expression. METHODS: HRPE cells were stimulated for 2, 4, 8, or 24 hours with 20% conditioned media (CM) from T-lymphocytes stimulated with CD3 or CD28 monoclonal antibodies (mAbs) or phorbol myristic acid. In some experiments, CM from CD3 mAb-stimulated T-lymphocytes was preincubated with neutralizing anti-(alpha)-tumor necrosis factor (TNF), alpha-interferon-gamma (IFN-gamma), or alpha-interleukin-1 (IL-1) mAb (control) to determine the contributions of each of these cytokines to HRPE chemokine induction by stimulated T-lymphocyte CM. HRPE cells were stimulated for 8 and 24 hours with IL-1 beta (0.2 to 20.0 ng/ml) (positive control), TNF-alpha (0.2 to 20.0 ng/ml) (positive control), IFN-gamma (1 to 1000 U/ml), IFN-gamma + IL-1 beta, IFN-gamma + TNF-alpha. Interleukin-2 (IL-2; 100 ng/ml) alone or in combination with IL-1 beta, TNF-alpha, or IFN-gamma also was tested. Enzyme-linked immunosorbent assay (ELISA) and Northern blot analyses were performed to determine secreted IL-8 and MCP-1 and their steady state mRNA expression, respectively. RESULTS: ELISA showed significant increases in HRPE IL-8 and MCP-1 secretion by CM from T-lymphocytes stimulated with CD3 or CD3 + CD28 mAb. Smaller, but significant, increases in IL-8 and MCP-1 resulted from CM phorbol myristic acid-stimulated T-lymphocytes. CM preincubated with neutralizing alpha-TNF or alpha-IFN-gamma mAb induced significantly less HRPE IL-8 and MCP-1, whereas preincubation of CM with neutralizing alpha-IL-1 mAb failed to inhibit CM-induced IL-8 or MCP-1. Northern blot analysis showed increased HRPE IL-8 and MCP-1 mRNA expression within 2 hours of stimulation and was maintained up to 24 hours. CM from T-lymphocytes stimulated with CD3 mAb or CD3 + CD28 mAb produced the greatest increases in IL-8 and MCP-1 mRNA. IFN-gamma induced dose-dependent increases in HRPE MCP-1, but not IL-8, IFN-gamma potentiated IL-1 beta and TNF-alpha-induced MCP-1 production, but showed little modulation of IL-1 beta and TNF-alpha-induced IL-8 production. IL-2 did not induce HRPE IL-8 or MCP-1, nor did it modulate the effects of the other cytokines. Northern blot analysis confirmed the ELISA results. CONCLUSIONS: T-lymphocyte secretions induce HRPE IL-8 and MCP-1 gene expression and secretion. TNF and IFN-gamma appear to be necessary components of T-lymphocyte CM for the induction of HRPE IL-8 and MCP-1. IFN-gamma alone induces HRPE MCP-1, albeit to a lesser extent than would IL-1 beta or TNF-alpha, and potentiates IL-1 beta- and TNF-alpha-induced HRPE MCP-1. IL-2 does not appear to modulate cytokine-induced HRPE IL-8 or MCP-1.     

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.