Role of endogenous IFN-gamma in macrophage programming induced by IL-12 and IL-18.

Besides the established role of interleukin-12 (IL-12) and IL-18 on interferon-gamma (IFN-gamma) production by natural killer (NK), T, and B cells, the effects of these cytokines on macrophages are largely unknown. Here, we investigated the role of IL-12/IL-18 on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by CD11b(+) adherent peritoneal cells, focusing on the involvement of endogenously produced IFN-gamma. C57BL/6 cells released substantial amounts of NO when stimulated with IFN-gamma or lipopolysaccharide (LPS), but failed to respond to IL-12 or IL-18 or both. However, IL-12/IL-18 pretreatment was able to program these cells to release 6-8-fold more NO and TNF-alpha in response to LPS or Trypanosoma cruzi stimulation, with NO levels directly correlating with macrophage resistance to intracellular parasite growth. Analysis of IL-12/IL-18-primed cells from mice deficient in IFN-gamma, IFNGR, and IFN regulatory factor-1 (IRF-1) revealed that these molecules were essential for LPS-induced NO release, but TNF-alpha production was IFN-gamma independent. Conversely, the myeloid differentiation factor 88 (MyD88)-dependent pathway was indispensable for IL-12/IL-18-programmed LPS-induced TNF-alpha production, but not for NO release. Contaminant T and NK cells largely modulated the IL-12/IL-18 programming of LPS-induced NO response through IFN-gamma secretion. Nevertheless, a small population of IFN-gamma(+) cells with a macrophage phenotype was also identified, particularly in the peritoneum of chronically T. cruzi-infected mice, reinforcing the notion that macrophages can be an alternative source of IFN-gamma. Taken together, our data contribute to elucidate the molecular basis of the IL-12/IL-18 autocrine pathway of macrophage activation, showing that endogenous IFN-gamma plays an important role in programming the NO response, whereas the TNF-alpha response occurs through an IFN-gamma-independent pathway.     

Broadly impaired NK cell function in non-obese diabetic mice is partially restored by NK cell activation in vivo and by IL-12/IL-18 in vitro

NK cells represent a link between innate and adaptive immunity, and may play a role in regulating autoimmune disorders. We have characterized the NK cell population in non-obese diabetic (NOD) mice. The percentage and absolute numbers of NK cells were similar in NOD and control MHC-matched B6.g7 mice. However, the capacity of NOD NK cells to mediate natural cytotoxicity as well as FcR- and Ly49D-mediated killing was compromised in vitro, suggesting a defect affecting multiple activation pathways. The defect was neither linked to the NK gene complex nor to the MHC, as determined by comparison with mice congenic for these regions. Introducing the beta(2)-microglobulin mutation on the NOD background further impaired NK cell function, showing that the compromised cytotoxic capacity in these two strains arises from two independent mechanisms. In vivo rejection responses against tumor cells and against MHC class I-deficient spleen cells were decreased in naive NOD recipients, but restored in mice pre-activated with tilorone, a potent activator of NK cells. In addition, killing of some tumor targets was restored in vitro after activation of NK cells with IL-12 plus IL-18 or with IFN-alpha/beta, but not with IL-2. Interestingly, natural killing of RMA-S targets by NOD NK cells could not be restored in vitro, indicating that restoration of killing capacity was only partial. Our data suggest a severe, but partially restorable, killing defect in NOD NK cells, affecting activation through several pathways.     

Curcumin attenuates the expression of IL-1beta, IL-6, and TNF-alpha as well as cyclin E in TNF-alpha-treated HaCaT cells; NF-kappaB and MAPKs as potential upstream targets

TNF-alpha induces some proinflammatory cytokines including IL-1beta, IL-6, IL-8, and itself by activation of NF-kappaB or MAPKs (p38, JNK, ERK). These cytokines play important roles in various inflammatory skin diseases, such as psoriasis. Recently it was also reported that expression of cyclin E is up-regulated by ERK pathway after TNF-alpha treatment. However, it was unknown whether curcumin, showing inhibitory effects on NF-kappaB and MAPKs, attenuates the expression of TNF-alpha-induced IL-1beta, IL-6, IL-8, and TNF-alpha as well as cyclin E expression in HaCaT cells. In this study, we investigated the inhibitory effect of curcumin on expression of proinflammatory cytokines and cyclin E in TNF-alpha-treated HaCaT cells. We found that curcumin inhibited the expression of TNF-alpha-induced IL-1beta, IL-6, and TNF-alpha, but not IL-8, in TNF-alpha-treated HaCaT cells as well as the TNF-alpha-induced cyclin E expression. In addition, curcumin inhibited the activation of MAPKs (JNK, p38 MAPK, and ERK) and NF-kappaB in TNF-alpha-treated HaCaT cells. Taken together, curcumin exerts anti-inflammatory and growth inhibitory effects in TNF-alpha-treated HaCaT cells through inhibition of NF-kappaB and MAPK pathways.     

Production of TNF-alpha, IL-6 and TGF-beta, and expression of receptors for TNF-alpha and IL-6, during murine Mycobacterium avium infection.

The Mycobacterium avium complex comprises intracellular bacteria associated with disseminated infection in patients with acquired immune deficiency syndrome (AIDS). Immune defects that lead to infection are unknown but cytokines appear to play an important role in the immunomodulation of host defence mechanisms. We evaluated the cytokine profiles seen temporally after murine M. avium infection. Spleen cells were obtained from M. avium-infected C57BL/6 mice and uninfected mice at weeks 1, 2, 3, 4 and 5. Cells were cultured in vitro and subsequently pulsed with killed M. avium. Supernatants were collected from the cultured splenic cells and the concentrations of interleukin-6 (IL-6), transforming growth factor-beta 1 (TGF-beta 1) and tumour necrosis factor-alpha (TNF-alpha) were measured. TGF-beta 1 was detected at week 1, followed by IL-6 production at week 2. Elevated TNF-alpha levels were observed at week 3. The addition of polyclonal anti-TGF-beta 1 antibody to M. avium-infected peritoneal macrophages in the presence of splenic cell supernatants from weeks 1, 3 and 5 led to decreased bacterial counts compared to controls. Anti-IL-6 antibody did not have any effect on macrophage anti-mycobacterial activity. Concurrently, we observed decreased expression of TNF-alpha receptors on infected macrophages. We propose that the early elevated levels of TGF-beta 1, a known suppressor of macrophage function, in conjunction with down-regulation of TNF-alpha receptors may help explain the suboptimal macrophage response to TNF-alpha, leading to impaired anti-mycobacterial activity.     

IL-15 and GM-CSF stimulated macrophages enhances phagocytic activity in ENU induced leukemic mice

Murine splenic macrophage plays a decisive role in host immunity through phagocytosis against pathogens. It was reported that, macrophages also involves in phagocytosis of some tumour cells upon its activation initiated by certain cytokines produced by other immune cell or by indigenously treated. In this study, we have investigated the killing of leukemic blast cells by macrophages upon stimulated with IL-15 and GM-CSF alone or in combination in ENU challenged leukemic murine model. Along with, the release of TNF-α, IL-12 and IFN-γ by macrophages were assayed by ELISA. NO production by macrophages was also investigated. The molecular expressions like GM-CSF and TLRs were investigated for better understand of macrophage-leukemic cell interaction. Result shows that in disease condition macrophages have poor phagocytic activities which may be due to less release of TNF-α, IL-12 and IFN-γ by macrophages. This impaired phagocytic activity in leukemic mice was increase upon stimulation with IL-15 and GM-CSF.     

Proinflammatory cytokines induce lymphocyte apoptosis in acute African swine fever infection

Twenty-one pigs inoculated with a highly virulent isolate (E70) of African swine fever (ASF) virus were killed 1-7 days later; a further three animals served as uninfected controls. An early increase in TNF-alpha, IL-1alpha, IL-1beta and IL-6 expression was detected in lymphoid organs from infected animals, together with an increase in the serum concentrations of TNF-alpha and IL-1beta. These changes were accompanied by increased apoptosis of lymphocytes, and the presence of infected and uninfected macrophages showing changes indicative of secretory and phagocytic activation. The present study demonstrated an increase in the number of macrophages expressing TNF-alpha, IL-1 and IL-6 in proximity to lymphocytes undergoing apoptosis, supporting previous suggestions that in acute ASF proinflammatory cytokines induce lymphocyte apoptosis.     

Heterogeneity of kupffer cells and splenic,alveolar, and peritoneal macrophages for the production of TNF,IL-1, AND IL-6

Kupffer cells and alveolar, splenic, and peritoneal macrophages from normal rats were incubated for various periods of time in the presence of LPS, and the culture supernatants were analyzed for IL-6, IL-1, and TNF. There was very little difference in the amounts of the cytokines produced by the macrophages when stimulated with 0.01–10/ml of LPS. The shapes of the time course curves for the production of the cytokines by the different types of macrophages were generally similar, although only Kupffer cells continued to produce IL-6 throughout the entire incubation period and splenic macrophages showed a lag period in the production of IL-1. Kupffer cells produced more IL-6 than that produced by the other populations of macrophages, and alveolar macrophages produced more IL-1 compared to that produced by splenic cells. Kupffer cells and peritoneal macrophages produced more IL-6 in 24 h than in 6 h of culture, and splenic macrophages produced more IL-1 in 24 compared to 6 h of culture. Alveolar macrophages produced more TNF than that produced by the other populations of cells but only when integrated over the entire incubation period. These results confirm and extend the observed functional heterogeneity of macrophages obtained from different tissues of the same animal. This study and future studies will lead to a better understanding of the role of cytokines in the inflammatory response.     

Corticotropin-releasing hormone augments proinflammatory cytokine production from macrophages in vitro and in lipopolysaccharide-induced endotoxin shock in mice

Corticotropin-releasing hormone (CRH) exerts an anti-inflammatory effect indirectly, via cortisole production, and a proinflammatory effect directly on immune cells. The aim of the present work was to examine the effect of CRH on macrophage-derived cytokines both in vitro and in vivo. For the in vitro experiments we used two types of macrophages: (i) the RAW264.7 monocyte/macrophage cell line and (ii) thioglycolate-elicited peritoneal macrophages from BALB/c mice. We have found that CRH enhanced lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 production. For the in vivo experiments we have used the LPS-induced endotoxin shock model in BALB/c mice, an established model for systemic inflammation in which macrophages are the major source of the proinflammatory cytokines responsible for the development of the shock. Administration of antalarmin, a synthetic CRH receptor 1 (CRHR1) antagonist, prior to LPS prolonged survival in a statistically significant manner. The effect was more evident at the early stages of endotoxin shock. CRHR1 blockade suppressed LPS-induced elevation of the macrophage-derived cytokines TNF-alpha, IL-1beta, and IL-6, confirming the role of CRH signals in cytokine expression. In conclusion, our data suggest that CRH signals play an early and crucial role in augmenting LPS-induced proinflammatory cytokine production by macrophages. Our data suggest that the diffuse neuroendocrine system via CRH directly affects the immune system at the level of macrophage activation and cytokine production.     

Preexposure of murine macrophages to CpG oligonucleotide results in a biphasic tumor necrosis factor alpha response to subsequent lipopolysaccharide challenge

Bacterial DNA and synthetic oligonucleotides containing CpG sequences (CpG-DNA and CpG-ODN) provoke a proinflammatory cytokine response (tumor necrosis factor alpha [TNF-alpha], interleukin-12 [IL-12], and IL-6) and increased mortality in lipopolysaccharide (LPS)-challenged mice via a TNF-alpha-mediated mechanism. It was hypothesized that preexposure of macrophages to CpG-ODN would result in an increased TNF-alpha response to subsequent LPS challenge in vitro. Using the murine macrophage cell line RAW 264.7, we demonstrated both a rapid proinflammatory cytokine response (TNF-alpha) and a delayed inhibitory cytokine response (IL-10) with CpG-ODN. Preexposure of macrophages to CpG-ODN for brief periods (1 to 3 h) augmented TNF-alpha secretion and mRNA accumulation following subsequent LPS challenge (1 microg/ml). However, prolonged preexposure to CpG-ODN (6 to 9 h) resulted in suppression of the TNF-alpha protein and mRNA response to LPS. The addition of anti-IL-10 antibody to CpG-ODN during preexposure resulted in an increase in the LPS-induced TNF-alpha response over that induced by CpG-ODN preexposure alone. Thus, while brief preexposure of macrophages to CpG-ODN augments the proinflammatory cytokine response to subsequent LPS challenge, prolonged preexposure elicits IL-10 production, which inhibits the TNF-alpha response. Although the initial proinflammatory effects of CpG-DNA are well established, the immune response to CpG-DNA may also include autocrine or paracrine feedback mechanisms, leading to a complex interaction of proinflammatory and inhibitory cytokines.     

Tumor necrosis factor alpha upregulates human microglial cell production of interleukin-10 in vitro.

Interleukin (IL)-10 appears to play an important regulatory role in the systemic inflammatory response; however, production of IL-10 within the human central nervous system has not been described. Using cultures of human fetal microglial cells, the resident macrophages of the brain, we investigated the production and regulation of bioactive IL-10. Lipopolysaccharide stimulated acute release of tumor necrosis factor (TNF)-alpha (peak by 8 h) and delayed production of IL-10 (over a 48-h period) in microglial cell cultures. Treatment of microglial cell cultures with TNF-alpha and IL-6 resulted in a dose-dependent release of IL-10. These cytokines also induced expression of IL-10 mRNA. Treatment of microglial cell cultures with IL-10 markedly inhibited TNF-alpha and IL-6 production. These findings suggest that during inflammation within the brain, acute release of TNF-alpha and IL-6 by activated microglia could promote subsequent release of IL-10, which functions to minimize the potential neurotoxic effects of proinflammatory cytokines.     

IL-12 directly stimulates expression of IL-10 by CD5+ B cells and IL-6 by both CD5+ and CD5- B cells: possible involvement in age-associated cytokine dysregulation

Constitutive expression of p35 and p40 IL-12 mRNA was detected in splenic macrophages isolated from aged mice. Macrophages were also implicated as the cell type responsible for the dysregulated IL-6 and tumor necrosis factor (TNF)-alpha commonly observed to be constitutively produced by lymphoid cells from aged donors. A role for IL-12 in the aging process was suggested when it was found that recombinant IL-12 (rIL-12) directly stimulated splenic CD5+ B cells to secrete IL-10, and both CD5+ and CD5- B cells could be directly induced to produce IL-6 in response to rIL-12. Furthermore, splenocytes from aged animals cultured in the presence of anti-IL-12 antibodies demonstrated a significant reduction in spontaneous IL-6, IL-10 and IFN- gamma production. Based on these observations it was concluded that IL- 12 might be responsible for the dysregulated production of IL-10 and IFN-gamma known to occur in aged animals. Treatment of aged animals with low doses of dehydroepiandrosterone sulfate, previously established to be immunocorrective in immunosenescent animals, reduced the age-associated alterations in IL-12 mRNA and protein expression. The mechanisms responsible for the abnormal constitutive expression of inflammatory cytokines by the macrophages of aged animals may play an important afferent role in establishing the immunosenescent phenotype.      

Modulation of cell-mediated immune response in B16F-10 melanoma-induced metastatic tumor-bearing C57BL/6 mice by sulforaphane

Effect of sulforaphane on cell-mediated immune response (CMI) was studied in B16F-10 melanoma-induced metastasis-bearing C57BL/6 mice. Administration of sulforaphane significantly enhanced natural killer (NK) cell activity in metastatic tumor-bearing animals (43.17% cell lysis, on day 5) and the activity was observed earlier than in tumor-bearing control animals (maximum of 9.76% cell lysis, on day 9). Antibody-dependent cellular cytotoxicity also was enhanced significantly in metastatic tumor-bearing animals (41.20% cell lysis on day 9) after sulforaphane administration compared with untreated control tumor-bearing animals (maximum of 12.62% cell lysis on day 15). An early antibody-dependent complement-mediated cytotoxicity also was observed in sulforaphane-treated tumor-bearing animals (26% cell lysis, on day 15). Administration of sulforaphane significantly enhanced the production of IL-2 and IFN-gamma in metastatic tumor-bearing animals. In addition, sulforaphane significantly downregulated the serum levels of proinflammatory cytokines such as IL-1beta, IL-6, TNF-alpha, and GM-CSF during metastasis. These data clearly suggest that sulforaphane effectively inhibited the spread of metastatic tumor cells through the stimulation of CMI, upregulation of IL-2 and IFN-gamma, and downregulation of proinflammatory cytokines IL-1beta, IL-6, TNF-alpha, and GM-CSF.     

Interaction of IL-1 beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) in human T cells activated by murine antigens.

We used a mixed leucocyte culture between human T cells and irradiated murine splenocytes which allowed us to distinguish between cytokine production from the responder and stimulator cells by the use of species-specific assays for mRNA up-regulation. Using this model of T cell activation by antigen, we studied the effects of human antigen-presenting cell-derived cytokines IL-1 beta, IL-6 and TNF-alpha on the activation of human T cell subsets. We show in this system that exogenously added IL-1 beta, IL-6 and TNF-alpha induces IL-2 receptor (R) up-regulation and IL-2 production, and proliferation by both CD4+ and CD8+ cells. The addition of IL-1 beta induces IL-6 mRNA, and anti-IL-1 antibodies or an IL-1R antagonist protein completely suppresses IL-6 and TNF-alpha supported proliferation. Similarly, addition of IL-6 or TNF-alpha induces up-regulation of IL-1 beta mRNA. However, anti-IL-6 and anti-IL-6R antibodies only partially block proliferation supported by IL-1 beta. These findings suggest that IL-6 and TNF-alpha will induce IL-2R up-regulation/IL-2 secretion via the induction of IL-1 beta production.     

Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.

Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.     

CD160: a unique activating NK cell receptor

Here we discuss CD160 an essential NK cell activating receptor that remains poorly understood. CD160 receptor exhibits a number of unique structural and functional characteristics that are not common to other killer immunoglobulin-like receptors that recognize major histocompatibility complex (MHC) class I molecules: (1) In addition to humans and mice, the cd160 gene is conserved in several other mammal species; (2) cd160 is located outside the NK gene complex and the Leukocyte Receptor Complex in humans; (3) CD160 expression is associated to the CD56(dim) CD16+ cytotoxic NK cell phenotype; (4) both human and mouse CD160 recognize MHC class Ia and Ib molecules; (5) unlike the other MHC class I-dependent activating NK receptors, CD160 is a glycosylphosphatidylinositol-anchored molecule with a single immunoglobulin-like domain, and does not bear immunoreceptor tyrosine-based activation motifs. Consequently, CD160 cannot signal by itself, requiring the recruitment of adaptor proteins. CD160 recruits phosphoinositide-3 kinase to trigger cytotoxicity and cytokine secretion; (6) specific engagement of NK CD160 receptor expressed by circulating NK cells produces proinflammatory cytokines IFN-γ, TNF-α, and, most notably, IL-6 and IL-8 as well as MIP1-β chemokine. The level of CD160-mediated IFN-γ production is always higher than the one observed after engagement of the CD16 receptor.     

Ly108 expression distinguishes subsets of invariant NKT cells that help autoantibody production and secrete IL-21 from those that secrete IL-17 in lupus prone NZB/W mice

Lupus is a systemic autoimmune disease characterized by anti-nuclear antibodies in humans and genetically susceptible NZB/W mice that can cause immune complex glomerulonephritis. T cells contribute to lupus pathogenesis by secreting pro-inflammatory cytokines such as IL-17, and by interacting with B cells and secreting helper factors such as IL-21 that promote production of IgG autoantibodies. In the current study, we determined whether purified NKT cells or far more numerous conventional non-NKT cells in the spleen of NZB/W female mice secrete IL-17 and/or IL-21 after TCR activation in vitro, and provide help for spontaneous IgG autoantibody production by purified splenic CD19⁺ B cells. Whereas invariant NKT cells secreted large amounts of IL-17 and IL-21, and helped B cells, non-NKT cells did not. The subset of IL-17 secreting NZB/W NKT cells expressed the Ly108^loCD4⁻NK1.1⁻ phenotype, whereas the IL-21 secreting subset expressed the Ly108^hiCD4⁺NK1.1⁻ phenotype and helped B cells secrete a variety of IgG anti-nuclear antibodies. α-galactocylceramide enhanced the helper activity of NZB/W and B6.Sle1b NKT cells for IgG autoantibody secretion by syngeneic B cells. In conclusion, different subsets of iNKT cells from mice with genetic susceptibility to lupus can contribute to pathogenesis by secreting pro-inflammatory cytokines and helping autoantibody production.     

IL-18-deficient mice are resistant to endotoxin-induced liver injury but highly susceptible to endotoxin shock

IL-18 is an IL-1-related cytokine which shares biological functions with IL-12. These include the activation of NK cells, induction of IFN-gamma production and Th1 cell differentiation. In this study we analyzed the effect of IL-18 deficiency on lipopolysaccharide (LPS)-induced liver injury and endotoxin shock in Propionibacterium acnes-primed mice. P. acnes-primed IL-18-deficient (IL-18KO) mice showed resistance to LPS-induced liver injury. Unexpectedly, P. acnes-primed IL-18KO mice were highly susceptible to LPS-induced endotoxin shock. Serum level of tumor necrosis factor (TNF)-alpha were markedly elevated (approximately 10-fold higher) within 1.5 h after LPS challenge in IL-18KO mice as compared with wild-type mice. Anti-TNF-alpha antibody administration to IL-18KO mice was significantly protective against endotoxin-induced lethality. P. acnes-primed IL-18KO macrophages produced approximately 6-fold more TNF-alpha protein than did P. acnes-primed wild-type control macrophages. Taken together, these findings demonstrate that IL-18 is responsible for the progression of endotoxin-induced liver injury as well as down-regulation of endotoxin-induced TNF-alpha production in P. acnes-primed mice.