The Fas death pathway controls coordinated expansions of type 1 CD8 and type 2 CD4 T cells in Trypanosoma cruzi infection

We investigated the role of the Fas ligand (FasL)/Fas death pathway on apoptosis and cytokine production by T cells in Trypanosoma cruzi infection. Anti-FasL, but not anti-TNF-alpha or anti-TRAIL, blocked activation-induced cell death of CD8 T cells and increased secretion of IL-10 and IL-4 by CD4 T cells from T. cruzi-infected mice. CD4 and CD8 T cells up-regulated Fas/FasL expression during T. cruzi infection. However, Fas expression increased earlier in CD8 T cells, and a higher proportion of CD8 T cells was activated and expressed IFN-gamma compared with CD4 T cells. Injection of anti-FasL in infected mice reduced parasitemia and CD8 T cell apoptosis and increased the ratio of CD8:CD4 T cells recovered from spleen and peritoneum. FasL blockade increased the number of activated T cells, enhanced NO production, and reduced parasite loads in peritoneal macrophages. Injection of anti-FasL increased IFN-gamma secretion by splenocytes responding to T. cruzi antigens but also exacerbated production of type 2 cytokines IL-10 and IL-4 at a late stage of acute infection. These results indicate that the FasL/Fas death pathway regulates apoptosis and coordinated cytokine responses by type 1 CD8 and type 2 CD4 T cells in T. cruzi infection.     

IL-4 promotes the formation of multinucleated giant cells from macrophage precursors by a STAT6-dependent, homotypic mechanism: contribution of E-cadherin

Multinucleated giant cells (MNG) are central players in the inflammatory response to foreign materials and in adverse responses to implants. IL-4 promotes the formation of MNG from bone marrow-derived precursors in vitro and participates in the development of the foreign body reaction in vivo. Therefore, we investigated the mechanism by which IL-4 promotes formation of MNG and engulfment of foreign bodies. We found that generation of MNG cells by IL-4 was dependent on cell density and expression of STAT6; macrophages derived from STAT6(-/-) mice were unable to form MNG in response to IL-4. No soluble factors including CCL2 or supernatants from IL-4-treated macrophages compensated for the lack of MNG cells in STAT6(-/-) cultures. We found that IL-4 must remain present during the full differentiation process and that STAT6(+/+) macrophage precursors retained their ability to differentiate into MNG over time. These MNG were able to internalize large particles efficiently, and the mononuclear STAT6(-/-) macrophages were unable to do so. Furthermore, we found that IL-4 induced expression of E-cadherin and dendritic cell-specific transmembrane protein in a STAT6-dependent manner. E-cadherin expression was critical for the formation of MNG cells by IL-4; an anti-E-cadherin antibody prevented the formation of large MNG. In addition, we found that STAT6(-/-) progenitors failed to fuse with STAT6(+/+), revealing the need for a homotypic interaction. Thus, IL-4 promotes the formation of MNG in a STAT6-dependent manner by regulating cell surface expression of E-cadherin, leading to homotypic cell fusion and the incorporation of large foreign bodies.     

Activated peritoneal macrophages inhibit the proliferation of rat ascites hepatoma AH-130 cells via the production of tumor necrosis factor-<Emphasis Type="Italic">α</Emphasis> and nitric oxide

OBJECTIVE: To study the effect of peritoneal macrophages on tumor cell proliferation, we cultured ascites hepatoma AH-130 cells with unstimulated, or lipopolysaccharide (LPS)- or interleukin (IL)-2-stimulated rat peritoneal macrophages, and examined the proliferation of AH-130 cells. MATERIALS AND METHODS: Rat peritoneal macrophages isolated from male Wistar rats were co-cultured with AH-130 cells in the absence or presence of LPS or IL-2. After incubation, proliferation of AH-130 cells was analyzed using flow cytometry. In addition, the levels of tumor necrosis factor (TNF)-alpha and nitric oxide (NOx, nitrate + nitrite) in the culture supernatants were measured. Furthermore, anti-TNF-alpha antibody (10 microg/ml) and nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 100 microM) were added to the coculture, and their effect on AH-130 cell proliferation was examined. RESULTS: When AH-130 cells were co-cultured with unstimulated peritoneal macrophages, proliferation of AH-130 cells was not affected. In contrast, when AH-130 cells were cocultured with peritoneal macrophages in the presence of LPS (0.1-20 microg/ml) or IL-2 (1-200 U/ml), proliferation of AH130 cells was dose-dependently suppressed by LPS or IL-2. Moreover, LPS- or IL-2-stimulation increased the levels of TNF-alpha and NOx in the supernatants of AH-130 cell and macrophage co-culture, although LPS and IL-2 did not induce TNF-alpha and NOx production by AH-130 cells incubated without macrophages. Interestingly, anti-TNF-alpha antibody and L-NMMA significantly inhibited the suppression of AH-130 cell proliferation by LPS- or IL-2-stimulated macrophages (p < 0.05). Furthermore, exogenously added recombinant rat TNF-alpha (0.26-1300 ng/ml) or NO donor (GSNO, S-nitroso-L-glutathione) (0.1 - 10 mM) dose-dependently suppressed the proliferation of AH-130 cells in the absence of macrophages. CONCLUSION: Together these observations suggest that when peritoneal macrophages are activated by LPS and IL-2, they suppress the proliferation of ascites hepatoma AH-130 cells via the production of TNF-alpha and nitric oxide.     

Xid mouse lymphocytes respond to TI-2 antigens when co-stimulated by TI-1 antigens or lymphokines

Spleen cells from male (CBA/N x DBA/2) F1 hybrid mice do not significantly respond to in vitro stimulation by trinitrophenyl-conjugated polyacrylamide beads (TNP-PAA), whereas the same antigen elicits high PFC responses in female F1 hybrid cells. Therefore, this antigen could be classified as a T-independent type 2 (TI-2) antigen. When male spleen cells were co-stimulated by TNP-PAA and TI type 1 antigen, either LPS or Brucella abortus, they produced vigorous anti-TNP responses. A similar increase of the in vitro responsiveness of male F1 hybrid spleen cells to TNP-PAA antigen was provoked by the addition of supernatants from P 388-D1 cells stimulated by muramyl-dipeptide (MDP) mainly containing interleukin-1 (IL-1) or supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated EL-4 cells that contained T-cell factors. The PFC response to another TI-2 antigen, TNP-Ficoll, was also significantly enhanced after co-stimulation by P 388-D1 supernatants. The response to TI-2 antigens being macrophage dependent, the influence of supernatants of peritoneal macrophages from male and female F1 hybrids incubated with TNP-Ficoll on the PFC response of normal DBA/2 mouse spleen cells to sheep erythrocytes was assessed. It was found that macrophage supernatants from female hybrids regularly increased by more than two times this anti-SRBC PFC response, whereas macrophage supernatants from male F1 hybrids did not. Moreover, in a specific proliferation test measuring IL-1 activity, when macrophage supernatants from female F1 produced a 13-fold increase of thymidine incorporation, supernatants from male F1 only produced a three-fold increase. It is concluded that, in addition to the known defects of B cells from Xid mice, their macrophages are also defective.     

Macrophage depletion decreases IgG anti-DNA in cultures from (NZB x NZW)F1 spleen cells by eliminating the main source of IL-6.

We have studied the role of macrophages in the production of IgG anti-DNA autoantibodies by (NZB x NZW)F1 mice (B/W). One of the main features of the systemic lupus erythematosus (SLE)-like disease that affects these mice, is the presence of circulating IgG autoantibodies and immune complexes, which lead to renal failure and death by the age of 8-9 months. IgG autoantibodies are produced without in vitro stimulation by total spleen cells from these mice when they reach the age of 6 months. We have demonstrated that IL-6 increases the production of IgG autoantibodies in cultures of splenic purified B cells from the old B/W mice. The aim of this study was to show the involvement of macrophages in the production of IL-6 and consequently in the production of IgG anti-DNA antibodies in vitro. We show that elimination of the macrophages by different treatments led to reduction of the content of IL-6 in the supernatants as well as of IgG anti-DNA autoantibodies. Addition of fresh, splenic or peritoneal macrophages restored the production of autoantibodies in macrophage-depleted cultures from old B/W mice. There were no differences in the capacity of IL-6 production between macrophages from old or young B/W mice, but an important difference was observed between peritoneal and splenic macrophages, where the former produced much higher levels of IL-6, and consequently were more potent inducers of IgG autoantibodies. The present results reinforce the role of macrophages and IL-6 in the production of IgG anti-DNA autoantibodies in B/W mice. The implications of these results in the pathogenesis of the disease are discussed.     

Histamine- and serotonin-releasing activity of the supernatants from guinea pig spleen cell cultures

The action of supernatants from cultivated in vitro guinea pig spleen cells on the mast cells of guinea pigs, rats and hamsters was studied. It was found that supernatants from guinea pig spleen cell cultures are potent to release histamine from mast cells of the examined populations in a dose-dependent fashion. Histamine release from heterologous mast cells (especially from rat pleural mast cells) was significantly higher than that from homologous mesenteric mast cells. It was also demonstrated (on rat mast cells) that guinea pig spleen cell supernatants possessed not only histamine but 5-hydroxytryptamine releasing activity as well. Rat pleural mast cells were more sensitive and released more serotonin after challenge with spleen cell supernatants than peritoneal cells.     

Cytokine production by murine cells activated by erythrogenic toxin type A superantigen of Streptococcus pyogenes.

The mode of pathogenic action of the Steptococcus pyogenes superantigen erythrogenic toxin type A (ETA) in causing toxic shock-like syndrome in humans is thought to be mediated by massive release of cytokines by patients immune cells. The cytokine-inducing capacity of ETA as an extracellular protein was compared with that of lipopolysaccharide (LPS), a component of cell wall of gram-negative bacteria. Peritoneal macrophages and splenocytes of BALB/c and C3H/HeJ mice were stimulated by ETA and LPS. Tumor necrosis factor (TNF), interleukin 3 (IL-3) and interleukin 6 (IL-6) activities in the supernatants of stimulated cells were evaluated. In contrast to LPS, ETA induced only low amounts of IL-6 and no detectable TNF activities in peritoneal macrophage supernatants. ETA-triggered BALB/c and C3H/HeJ splenocytes produced great amounts of IL-6. ETA triggered the production of IL-3 by both mice strains splenocytes in a dose dependent manner. The amounts of IL-3 in supernatants were comparable to those induced by concanavalin A. The simultaneous presence of ETA and LPS in macrophage and splenocyte cultures induced a slight enhancement above an additive value after 72-96 h. Challenge of BALB/c mice with ETA 6 h before the harvest of peritoneal macrophages led to an enhanced production of IL-6 upon stimulation with ETA as well as with LPS. Splenocytes of nude BALB/c mice did not produce IL-6 upon stimulation with ETA, whereas LPS-induced IL-6 production was similar in these mice and in their littermates. The pathogenic effect of ETA on host's immune cells could most likely be explained as a consequence of T cell activation. The results confirm also that LPS- and ETA-induced shock is mediated by different cell types.     

Neutrophil Migration Induced by IL-1β Depends upon LTB4 Released by Macrophages and upon TNF-α and IL-1β Released by Mast Cells

In the present study, we investigate whether mast cells and macrophages are involved in the control of IL-1β-induced neutrophil migration, as well as the participation of chemotactic mediators. IL-1β induced a dose-dependent neutrophil migration to the peritoneal cavity of rats which depends on LTB₄, PAF and cytokines, since the animal treatment with inhibitors of these mediators (MK 886, PCA 4248 and dexamethasone respectively) inhibited IL-1β-induced neutrophil migration. The neutrophil migration induced by IL-1β is dependent on mast cells and macrophages, since depletion of mast cells reduced the process whereas the increase of macrophage population enhanced the migration. Moreover, mast cells or macrophages stimulated with IL-1β released a neutrophil chemotactic factor, which mimicked the neutrophil migration induced by IL-1β. The chemotactic activity of the supernatant of IL-1β-stimulated macrophages is due to the presence of LTB₄, since MK 886 inhibited its release. Moreover, the chemotactic activity of IL-1β-stimulated mast cells supernatant is due to the presence of IL-1β and TNF-α, since antibodies against these cytokines inhibited its activity. Furthermore, significant amounts of these cytokines were detected in the supernatant. In conclusion, our results suggest that neutrophil migration induced by IL-1β depends upon LTB₄ released by macrophages and upon IL-1β and TNFα released by mast cells.     

Studies of macrophage function in murine systemic lupus erythematosus. 4. Failure to reverse the defect in Fc-mediated phagocytosis and binding by in vitro stimulants or prostaglandins

Previous studies have shown that resident peritoneal macrophages from mice with systemic lupus erythematosus (SLE) show defective Fc-mediated phagocytosis and binding of opsonized sheep erythrocytes (EA) in vitro. Possible causes of this defect in (NZB X NZW)F1 (B/W) mice were investigated. These included a maturational block in peritoneal macrophage differentiation, the production by peritoneal cells of a factor which inhibits Fc receptor expression and phagocytosis, and an abnormal response by macrophages of autoimmune mice to prostaglandins. Resident peritoneal macrophages of B/W mice did not show a maturational block since incubation with either (a) differentiating agents such as 4 beta-phorbol 12 beta-myristate 13 alpha acetate or retinoic acid, or (b) lymphokine (LK), prepared by Con A stimulation of mouse spleen cells, failed to enhance Fc-mediated phagocytosis and binding by B/W cells relative to controls. However, LK from B/W and B6AF1 cells stimulated Fc-mediated phagocytosis and binding by bone-marrow (BM)-derived macrophages of CBA/H mice; B/W LK also stimulated BM cells from B/W mice. Peritoneal cell supernatants did not inhibit phagocytosis of Fc receptor expression by BM-derived macrophages in vitro. Prostaglandin E treatment of peritoneal or BM-derived macrophages in vitro failed to restore decreased phagocytosis and binding of EA induced by culture in indomethacin and failed to stimulate phagocytosis by untreated cultures. The reason for the observed defect remains obscure.     

Further observations on the immunological induction of DNA synthesis in mouse peritoneal macrophages. Role of products of activated lymphocytes.

Subcutaneous injection of ovalbumin (OA) into mice immunized with OA in Freund's complete adjuvant was followed by an increase in the numbers of peritoneal macrophages synthesizing DNA, determined by autoradiography. The effect was immunologically specific. The increase was followed by an increase in the numbers of peritoneal macrophages; the numbers of peritoneal lymphocytes also increased. Injection of OA into immunized or normal mice was followed by a blood monocytosis. Increased DNA synthesis, determined by liquid scintillation counting, occurred in spleen or lymph node cells from immunized mice, cultured with OA. Diluted supernatants from such cultures, injected intravenously into normal mice, caused increases in the numbers of DNA-synthesizing peritoneal macrophages. Similarly, supernatants from concanavalin A stimulated spleen cells, freed of Con A, also caused an increase in DNA-synthesizing macrophages.     

Protein malnutrition and the febrile response in the Fischer rat

We assessed the effect of protein deprivation on the ability of peritoneal macrophages from Fischer rats to produce interleukin-1 (IL-1) after in vitro stimulation. Pyrogenic activity of supernatants was measured by an in vivo febrile response assay. Control rats were given a 23% casein diet and protein-malnourished rats were given an 8% casein diet for 4 weeks. IL-1-containing supernatants prepared from peritoneal macrophages were injected into assay rats, whose temperatures were measured for 6 hours (delta T6). Rats injected with IL-1-containing supernatants derived from peritoneal macrophage cultures of protein-deprived rats had significantly less fever (delta T6 = 0.20 +/- 0.09 degree C) than rats injected with IL-1 containing supernatants derived from peritoneal macrophage cultures of control rats (delta T6 = 0.56 +/- 0.09 degree C), P less than .01. Protein malnutrition leads to diminished pyrogenicity of macrophage culture supernatants and may be at least partly responsible for the decreased febrile response seen in the malnourished animals.     

Study of the biological activities of toxic shock syndrome toxin-1: II. Induction of the proliferative response and the interleukin 2 production by T cells from human peripheral blood mononuclear cells stimulated with the toxin

Toxic shock syndrome toxin-1 (TSST-1) is an exotoxin produced by Staphylococcus aureus isolated from patients with toxic shock syndrome. We investigated the proliferative response of human lymphocytes and their interleukin 2 (IL-2) production after stimulation with TSST-1 in vitro. Human cord blood mononuclear cells (HCBM) and human peripheral blood mononuclear cells (HPBM) could proliferate with TSST-1 stimulation. T cell-depleted HPBM showed only a marginal response to this toxin. A IL-2-like factor with a molecular weight of 15-18 kD was obtained from the supernatants of TSST-1-stimulated HPBM cultures. The factor was absorbed by CTLL-2 cells but not by T cell-depleted murine spleen cells, indicating that it is IL-2. HPBM are very sensitive to TSST-1: a low concentration of TSST-1 (0.01 ng/ml in 36 h stimulation) and a short period of stimulation (8 h at 10 ng/ml of the toxin) were fully effective for HPBM to produce substantial amounts of IL-2. Removal of T cells abrogated the TSST-1-induced IL-2 production by HPBM. Reconstituted cell cultures of nylon wool column-passed T cells and macrophages produced IL-2 by TSST-1 stimulation and, furthermore, the accessory activity of the macrophages could be partially replaced by a macrophage-derived factor containing interleukin 1. These findings indicate that T cells require macrophages or IL-1 for TSST-1-induced production of IL-2. The roles of lymphokines, including IL-2, in the development of this illness are discussed.     

FasL and TRAIL induce epidermal apoptosis and skin ulceration upon exposure to Leishmania major

Receptor-mediated apoptosis is proposed as an important regulator of keratinocyte homeostasis in human epidermis. We have previously reported that Fas/FasL interactions in epidermis are altered during cutaneous leishmaniasis (CL) and that keratinocyte death through apoptosis may play a pathogenic role for skin ulceration. To further investigate the alterations of apoptosis during CL, a keratinocyte cell line (HaCaT) and primary human epidermal keratinocytes were incubated with supernatants from Leishmania major-infected peripheral blood mononuclear cells. An apoptosis-specific microarray was used to assess mRNA expression in HaCaT cells exposed to supernatants derived from L. major-infected cultures. Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression were significantly up-regulated, and apoptosis was detected in both HaCaT and human epidermal keratinocyte cells. The keratinocyte apoptosis was partly inhibited through blocking of Fas or FasL and even more efficiently through TRAIL neutralization. Up-regulation of Fas on keratinocytes in epidermis and the presence of FasL-expressing macrophages and T cells in dermis were previously reported by us. In this study, keratinocytes expressing TRAIL, as well as the proapoptotic receptor TRAIL-R2, were detected in skin biopsies from CL cases. We propose that activation of Fas and TRAIL apoptosis pathways, in the presence of inflammatory mediators at the site of infection, leads to tissue destruction and ulceration during CL.     

Contribution of IL-18-induced innate T cell activation to airway inflammation with mucus hypersecretion and airway hyperresponsiveness

Human bronchial asthma is characterized by airway hyperresponsiveness (AHR), eosinophilic airway inflammation, mucus hypersecretion and high serum level of IgE. IL-18 was originally regarded to induce T(h)1-related cytokines from Th1 cells in the presence of IL-12. However, our previous reports clearly demonstrated that IL-18 with IL-2 promotes Th2 cytokines production from T cells and NK cells. Furthermore, IL-18 with IL-3 stimulates basophils and mast cells to produce Th2 cytokines. Thus, we examined the capacity of IL-2 and IL-18 to induce AHR, airway eosinophilic inflammation and goblet cell metaplasia. Intranasal administration of IL-2 and IL-18 induces AHR, mucus hypersecretion and eosinophilic inflammation in the lungs of naive mice. CD4+ T cells are prerequisite for this IL-2 plus IL-18-induced bronchial asthma, because CD4+ T cells-depleted or Rag-2-deficient (Rag-2-/-) mice did not develop bronchial asthma after IL-2 plus IL-18 treatment. Both STAT6-/- mice and IL-13-neutralized wild-type mice failed to develop AHR, goblet cell metaplasia and airway eosinophilic inflammation, while IL-4-/- mice almost normally developed, suggesting that IL-13 is a major causative factor and IL-4 mainly enhances the degree of AHR and eosinophilic inflammation. Both IL-4 and IL-13 equally induce eotaxin in mouse embryonic fibroblasts. However, only IL-13 blockade inhibited asthma symptoms, suggesting that IL-13 but not IL-4 is produced abundantly and plays a critical role in the pathogenesis of bronchial asthma in this model. As airway epithelial cells store robust IL-18, IL-18 might be critically involved in pathogen-induced bronchial asthma, in which pathogens stimulate epithelial cells to produce IL-18 without IL-12 induction.     

Interleukin-3 stabilizes CD124/IL-4α surface expression in mast cells via Tyk2 and STAT6

Interleukin (IL)-4 signals can modulate mast cells, which express the IL-4Rα chain. The IL-4Rα can heterodimerise with the common γ-chain and utilizes JAK1 and JAK2 for signal transduction, while complexes of IL-4Rα with IL-13Rα1 subunit mediates signals via JAK2 and Tyk2. Here, we report that IL-3 is an essential factor for the continuous expression of the IL-4Rα chain on mast cells, which did not express the IL-13Rα1 chain. We demonstrate that the signals induced by IL-3 important for IL-4Rα expression are mediated by Tyk2 and STAT6 activation and the subsequent maintenance of HSP90 levels. In line with that, inhibition of either Tyk2, STAT6 or HSP90 impaired the IL-3-induced IL-4Rα upregulation. Consequently, the IL-3 maintained IL-4Rα surface expression via Tyk2 is essential for the costimulatory effect of IL-4 on the IL-33-induced production of IL-6 and IL-13.     

Mechanisms of eosinophilia in mice infested with larval Haemaphysalis longicornis ticks.

Infestation of larval Haemaphysalis longicornis ticks induced a threefold increase of eosinophils in the peripheral blood of normal WBB6F1- +/+ mice 2 days after tick infestation. In genetically mast cell-deficient WBB6F1- W/Wv mice, a threefold increase of blood eosinophils was observed 6 days after the tick infestation. However, marked infiltration of eosinophils was detected in the tick infestation sites of the WBB6F1- +/+ mice but not the WBB6F1- W/Wv mice. When the mast cell deficiency of WBB6F1- W/Wv mice had been rescued locally by intradermal injections of WBB6F1- +/+ mouse-derived cultured mast cells, a rapid increase of blood eosinophils and tissue infiltration of eosinophils were revealed following tick infestation. The intravenous (i.v.) injection of immune spleen or lymph node cells obtained from WBB6F1- +/+ mice 10 days after tick infestation led to significant eosinophilia in naive recipient mice. Treatment with anti-Thy-1.2 or anti-CD4 monoclonal antibody (mAb) and complement (C) completely abolished the eosinophilia; the early response (2 days after tick challenge) is dependent on mast cells at the feeding site, and the late response (6 days after tick challenge) is dependent on T lymphocytes. Since amplified interleukin-5 (IL-5) cDNA was detectable in the spleen cells 4 days after tick infestation, the late response might be mediated by IL-5. The infiltration of eosinophils at the feeding site of skin appeared to be dependent on mast cells.     

FcgammaRIII-expressing macrophages are essential for development of collagen-induced arthritis

IgG-binding Fc receptors, and in particular FcgammaRIII, are crucial for induction of collagen-induced arthritis (CIA), as FcgammaRIII-deficient mice are highly protected to arthritis. However, which of the FcgammaRIII-expressing cells that is responsible for induction of arthritis is not known. In this study, we have addressed this question by purifying different FcgammaRIII(+) cell populations, transferred them to FcgammaRIII-deficient mice and studied if the recipient mice can develop arthritis. The cell populations were isolated from spleen, bone marrow and the peritoneal cavity. Our results show that FcgammaRIII(+) CD11b(+) peritoneal macrophages can render FcgammaRIII-deficient mice susceptible to CIA. In contrast, FcgammaRIII(-) peritoneal macrophages or FcgammaRIII(+) spleenocytes, bone marrow cells, mast cells or monocytes could not mediate this effect. To further evaluate the contribution of the FcgammaRIII(+) macrophages in arthritis, we investigated the cytokine profile in these cells during CIA. The arthritic macrophages exhibited significantly higher mRNA levels of TNFalpha and IL-12p35 compared with macrophages from normal mice. We conclude that FcgammaRIII-expressing macrophages, producing pro-inflammatory cytokine and T helper type 1 differentiating factor, are the major effector cells in the induction of CIA.