IL-10- and TGF-beta-mediated susceptibility in kala-azar and post-kala-azar dermal leishmaniasis: the significance of amphotericin B in the control of Leishmania donovani infection in India

Visceral leishmaniasis (VL) or kala-azar is known to be associated with a mixed Th1-Th2 response, and effective host defense requires the induction of IFN-gamma and IL-12. We address the role of the differential decline of IL-10 and TGF-beta in response to sodium antimony gluconate (SAG) and amphotericin B (AmB), the therapeutic success of SAG and AmB in Indian VL, and the significance of IL-10 and TGF-beta in the development and progression of post-kazla-azar dermal leishmaniasis (PKDL). In the active disease, PBMC from VL patients showed suppressed Ag-specific lymphoproliferation, IFN-gamma and IL-12 production, and elevation of IL-10 and TGF-beta. Cure corresponded with an elevation in IFN-gamma and IL-12 production and down-regulation of IL-10 and TGF-beta. Both CD4(+) and CD8(+) T cells were involved in IFN-gamma and IL-10 production. Interestingly, the retention and maintenance of residual IL-10 and TGF-beta in some SAG-treated individuals and the elevation of IL-10 and TGF-beta in PKDL, a sequel to kala-azar, probably reflects the role of these cytokines in reactivation of the disease in the form of PKDL. Contrastingly, AmB treatment of VL resulted in negligible TGF-beta levels and absolute elimination of IL-10, reflecting the better therapeutic activity of AmB and its probable role in the recent decline in PKDL occurrences in India. Moreover, elucidation of immune responses in Indian PKDL patients revealed a spectral pattern of disease progression where disease severity could be correlated inversely with lymphoproliferation and directly with TGF-beta, IL-10, and Ab production. In addition, the enhancement of CD4(+)CD25(+) T cells in active VL, their decline at cure, and reactivation in PKDL suggest their probable immunosuppressive role in these disease forms.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

Plasma levels of interleukin-12 (IL-12), interleukin-18 (IL-18) and transforming growth factor beta (TGF-beta) in Plasmodium falciparum malaria

The interaction between pro- and anti-inflammatory cytokines such as interleukin 12 (IL-12), interleukin 18 (IL-18) and transforming growth factor beta (TGF-beta) may play an important role in malaria pathogenesis and outcome. IL-18 cooperates with IL-12 in the IFN-gamma production by T, B, and NK cells, and synergizes with IL-12 for IFN-gamma production by Th1 cells. Recently it has been demonstrated that these cytokines modulate the immunoresponse in Plasmodium falciparum malaria. The aim of this study was to measure the plasma levels of IL-12, IL-18 and TGF-beta in 105 African children with various degrees of malaria, and correlate the production of these cytokines with the severity of the disease. IL-12, IL-18 and TGF-beta levels were determined using enzyme-linked immunosorbent assay. The severity of malaria was established by parasitemia, clinical symptoms and haematological parameters. The levels of IL-12, IL-18 and TGF-beta were found to be significantly elevated (15.6 + / - 12.3, 22.7 + / - 13.8 pg/ml and 25.14 + / - 13.22 pg/ml respectively) in all of the children. IL-12 and IL-18 levels were significantly lower (13.2 + / - 5.53 and 21.5 + / - 10 pg/ml pg/ml) in children with severe disease, whereas the level of TGF-beta was higher (28.09 + / - 12.39 pg/ml). In contrast, IL-12 and IL-18 levels were found to be higher (17.32 + / - 7.8 pg/ml and 25.7 + / - 7.6 pg/ml) in patients with mild disease, whereas the level of TGF-beta was lower (20.92 + / - 12.76 pg/ml) compared to the severe malaria group. The correlation between IL-12 and IL-18 demonstrated a progressive relationship up to a value of IL-12 < 25 pg/ml, while IL-18 remained stable at higher levels of IL-12. An inverse correlation was found between IL-12 and TGF-beta up to a value of IL-12 < 30, after which the level of TGF-beta remained stable. This finding suggests that fine mechanisms regulate the interaction between IL-12, IL-18 and TGF-beta in the immune response to Plasmodium falciparum.     

Parallel regulation of IL-4 and IL-5 in human helminth infections.

To investigate the relationship between cytokine production and the increased levels of serum IgE and peripheral eosinophilia commonly accompanying human helminth infections, we studied the ability of PBMC of normal (N1) (n = 18) and eosinophilic individuals with helminth infections (H1) (n = 9) to produce IL-3, IL-4, IL-5, granulocyte-macrophage-CSF, and IFN-gamma in vitro after stimulation with PMA (50 ng/ml) and ionomycin (1 microgram/ml). The two groups differed in both the levels of serum IgE and eosinophilia. For mitogen-induced production of granulocyte-macrophage-CSF and IFN-gamma, there was no difference in cytokine production between the two groups. In marked contrast, supernatants from PBMC of infected individuals had significantly higher levels of IL-4 (mean = 213 pg/ml for N1 and 944 pg/ml for H1, p less than 0.02), IL-5 (mean = 180 pg/ml for N1 and 1118 pg/ml for HL, p less than 0.001), and IL-3 (mean = 13900 pg/ml for N1, 28029 pg/ml for H1, p less than 0.05). In addition, helminth-infected patients had approximately 5-fold greater numbers of T cells capable of producing IL-5 and 2.5-fold greater frequency of IL-4-secreting cells than did normal individuals; GM-CSF- and IFN-gamma-producing T cell numbers were not significantly different in the two groups. IL-3-producing cell frequencies could not be evaluated by this method. There was a direct correlation between IL-4 production and IL-5 production at the level of both protein production and frequency of T cells capable of producing these cytokines. These data indicate that individuals with reactive eosinophilia and elevated serum IgE have an expanded population of lymphocytes producing IL-4 and IL-5 and the association of the two suggests that the regulation of IL-4 and IL-5 may be linked.     

Inhibition of inducible NO synthase by TH2 cytokines and TGF beta in rheumatoid arthritic synoviocytes: effects on nitrosothiol production.

The aim of this study was to compare the effects on NO production of IL-4, IL-10, and IL-13 with those of TGF-beta. RA synovial cells were stimulated for 24 h with IL-1 beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(-4)IU/ml) alone or in combination. Nitrite was determined by the Griess reaction, S-nitrosothiols by fluorescence, and inducible NO synthase (iNOS) by immunofluorescence and fluorescence activated cell sorter analysis (FACS). In other experiments, IL-4, IL-10, IL-13, and TGF beta were used at various concentrations and were added in combination with proinflammatory cytokines. The addition of IL-1 beta, TNF-alpha, and IFN-gamma together increased nitrite production: 257.5 +/- 35.8 % and S-nitrosothiol production : 413 +/- 29%, P < 0.001. None of these cytokines added alone had any significant effect. iNOS synthesis increased with NO production. IL-4, IL-10, IL-13, and TGF beta strongly decreased the NO production caused by the combination of IL-1 beta, TNF-alpha, and IFN-gamma. These results demonstrate that stimulated RA synoviocytes produce S-nitrosothiols, bioactive NO* compounds, in similar quantities to nitrite. IL-4, IL-10, IL-13, and TGF-beta decrease NO production by RA synovial cells. The anti-inflammatory properties of these cytokines may thus be due at least in part to their effect on NO metabolism.     

Soluble IL-2 receptor as an agent of serum-mediated suppression in human visceral leishmaniasis

In visceral leishmaniasis (VL), patient's lymphocytes are not activated by leishmania Ag stimulation, and their sera exhibit a potent nonspecific suppressive effect on the responses of normal lymphocytes. Sera were obtained from 33 VL patients, eight patients with subclinical VL, and from 27 normal volunteers. Only sera from VL patients markedly reduced Con A-induced lymphocyte proliferative responses, as well as IL-2 or IFN-gamma production by normal lymphocytes. Addition of exogenous human rIL-2 to cultures containing VL patient sera partially reversed the normal lymphocyte proliferative capacity and restored IFN-gamma production. This phenomenon was consistent with the presence of greatly elevated levels of soluble IL-2R (sIL-2R) in VL patients' sera (4299 +/- 2351 U/ml), well above those of normal sera (180 +/- 94 U/ml), or of sera from patients with subclinical leishmania infection without immunosuppression (1002 +/- 281 U/ml). Furthermore, the removal of sIL-2R reduced VL serum suppressive activity as evaluated by effects on IL-2 and on IFN-gamma production. These data suggest the participation of high levels of sIL-2R in the serum-mediated suppression in VL.     

TNF-alpha and IFN-gamma reverse IL-4 inhibition of lymphokine-activated killer cell function

Recombinant IL-4 inhibits IL-2-induced lymphokine-activated killer (LAK) cell development of PBMC. We evaluated the effect of various cytokines in reversing IL-4-mediated LAK inhibition. PBMC were cultured in IL-2 (10-1000 u/ml) with or without IL-4 (2-100 u/ml) and tested for cytotoxicity against the NK-sensitive K562 cells and NK-resistant UCLA-SO-M14 cells. Addition of IL-4 at the beginning of culture suppresses LAK activity in a dose-dependent fashion. Addition of IFN-gamma or TNF-alpha partially reverses IL-4-mediated inhibition (30-100%) in a dose-dependent fashion. IFN-gamma and TNF-alpha must be added within the first 24 hr of initiating culture in order to reverse IL-4 inhibition. Furthermore, IFN-gamma and TNF-alpha are most effective at reversing IL-4 inhibition at low concentrations of IL-2 (less than 100 u/ml). Addition of other IL-2-induced cytokines such as GM-CSF (50 u/ml), M-CSF (250 u/ml), and IFN-alpha (10-10,000 u/ml) fails to reverse IL-4 inhibition. In addition to suppression of LAK induction, IL-4 also inhibits IL-2-induced IFN-gamma and TNF-alpha protein production in PBMC. The reversal of IL-4-mediated LAK inhibition by TNF-alpha and IFN-gamma may therefore be due to resupply of these endogenously suppressed cytokines.     

IL-1 induces IL-1. III. Specific inhibition of IL-1 production by IFN-gamma

IL-1 possesses several biologic properties, some of which are associated with chronic inflammatory diseases. We have recently shown that IL-1 induces its own gene expression and, in the present studies, we have examined the effect of IFN-gamma on IL-1-induced IL-1 production. Whereas IFN-gamma increases the total amount of IL-1 (extracellular and cell-associated) produced after endotoxin stimulation of human PBMC, in the same cultures, IL-1-induced IL-1 production was markedly (greater than 70%) reduced in the presence of IFN-gamma. We observed this inhibition in the PBMC from over 40 human donors by employing non-cross-reacting RIA for either IL-1 beta or IL-1 alpha. IFN-gamma inhibited IL-1 beta-induced IL-1 alpha as well as IL-1 alpha-induced IL-1 beta production; furthermore, this inhibitory effect of IFN-gamma was unaffected by indomethacin. The ability of 100 U/ml of IFN-gamma to inhibit IL-1-induced IL-1 production was comparable to that accomplished by 10(-7) M dexamethasone. In contrast to its effect on IL-1 production from PBMC, IFN-gamma had no effect on the proliferative responses of T cells to IL-1. We conclude that IFN-gamma down-regulates synthesis of total IL-1-induced IL-1 production but up-regulates endotoxin-induced IL-1 production. These studies may explain the ameliorating effects of IFN-gamma in experimental models of IL-1-induced bone and cartilage degradation, in peritoneal fibrosis, and in patients with diseases associated with increased IL-1 production.     

Reduction in the number of UCHL-1+ cells and IL-2 production in the peripheral blood of patients with visceral leishmaniasis.

PBMC from patients with visceral leishmaniasis (VL), before and after successful antimony therapy, were analyzed for their phenotypes and for their ability to produce IL-2 and IFN-gamma and to proliferate against PHA and leishmanial Ag. In agreement with results of earlier studies, PBMC from active VL patients showed a markedly reduced proliferative response and IL-2 and IFN-gamma production, compared with those of healthy controls. The levels of CD4+ and CD8+ T cells were within the normal range, but there was a significant decrease in UCHL-1+ cells (helper-inducer), compared with healthy individuals. The inhibited cellular responses, and lymphokine secretion and decreased level of UCHL-1+ cells in the PBMC of the VL patients returned to the normal range after successful chemotherapy. PBMC from active VL patients were fractionated into adherent cells and nonadherent cells, and the non-adherent were further fractionated into UCHL-1+ and UCHL-1- subpopulations. Results from cell depletion and reconstitution experiments suggest that the IL-2 production by nonadherent cells stimulated with PHA was inhibited by adherent cells, but the IL-2 production by nonadherent cells in response to specific Ag was not. In contrast, UCHL-1- cells seem to mediate the inhibition of Ag-driven IL-2 production by nonadherent cells but not mitogen-stimulated IL-2 secretion by nonadherent cells. Ag-specific IL-2 production principally involves UCHL-1+ cells.     

Glycyrrhizin restores the impaired IL-12 production in thermally injured mice

Mice 6 days after thermal injury (TI-mice) did not respond to lipopolysaccharide (LPS) stimulation for production of serum interleukin 12 (IL-12; 2 h after LPS stimulation, <20 pg/ml in TI-mice; 1091+/-162 pg/ml in normal mice). However, 2 h after LPS stimulation, 1456+/-118 pg/ml of IL-12 were demonstrated in sera of TI-mice previously treated with a 10 mg/kg i.p. dose of glycyrrhizin (GR). IL-12 was not induced by LPS in sera of normal mice inoculated with burn-associated type 2 T cells (IL-4/IL-10-producing CD8+CD11b+TCRgamma/delta+T cells isolated from spleens of TI-mice). However, IL-12 production was induced by LPS in sera of these mice previously treated with GR or a mixture of monoclonal antibodies (mAbs) for type 2 cytokines. Also, IL-12 production was induced by LPS in TI-mice inoculated with CD4+T cells from spleens of GR-treated normal mice (GR-CD4+T cells, 5x10(6)cells/mouse). Since GR-CD4+T cells have been shown to be antagonistic cells against production of type 2 cytokines by burn-associated type 2 T cells, these results indicate that IL-12 unresponsiveness shown in TI-mice is recovered by GR through the regulation of burn-associated type 2 T cell responses.     

Soluble bacterial constituents down-regulate secretion of IL-12 in response to intact Gram-positive bacteria

Intact Gram-positive bacteria induce production of large amounts of IL-12 from freshly isolated human monocytes. Here the bacterial structures and signalling pathways involved were studied and compared with those leading to IL-6 production, and to IL-12 production in response to LPS after IFN-gamma pre-treatment. Intact bifidobacteria induced massive production of IL-12 (1 ng/ml) and IL-6 (>30 ng/ml) from human PBMC, whereas fragmented bifidobacteria induced IL-6, but no IL-12. IL-12 production induced by intact bifidobacteria was inhibited by pre-treatment with bifidobacterial sonicate, peptidoglycan, muramyl dipeptide, lipoteichoic acid, the soluble TLR2 agonist Pam(3)Cys-SK(4), or anti-TLR2 antibodies. Blocking of phagocytosis by cytochalasin, inhibition of the JNK or NF-kappaB pathways or treatment with Wortmannin also reduced the IL-12 response to intact Gram-positive bacteria. LPS induced moderate levels of IL-12 (0.31 ng/ml), but only from IFN-gamma pre-treated PBMC. This IL-12 production was enhanced by Wortmannin and unaffected by blocking the JNK pathway. Thus, intact Gram-positive bacteria trigger monocyte production of large amounts of IL-12 via a distinct pathway that is turned off by fragmented Gram-positive bacteria. This may be a physiological feedback, since such fragments may signal that further activation of the phagocyte via the IL-12/IFN-gamma loop is unnecessary.     

Selective suppression of IL-12 production by chemoattractants

We investigated the ability of chemoattractants to affect IL-12 production by human monocytes and dendritic cells. We found that pretreatment of monocytes with macrophage chemoattractant proteins (MCP-1 to -4), or C5a, but not stromal-derived factor-1, macrophage inflammatory protein-1alpha, RANTES, or eotaxin, inhibited IL-12 p70 production in response to stimulation with Staphylococcus aureus, Cowan strain 1 (SAC), and IFN-gamma. The production of TNF-alpha and IL-10, however, was minimally affected by any of the chemoattractants. The degree of inhibition of IL-12 p70 production by MCP-1 to -4 was donor dependent and was affected by the autocrine inhibitory effects of IL-10. In contrast, C5a profoundly suppressed IL-12 production in an IL-10-independent fashion. Neither TGF-beta1 nor PGE2 was important for the suppression of IL-12 by any of the chemoattractants tested. The accumulation of mRNA for both IL-12 p35 and p40 genes was inhibited by chemokine pretreatment. Interestingly, MCP-1 to -4 and C5a did not suppress IL-12 production by monocyte-derived dendritic cells (DC) stimulated with CD40 ligand and IFN-gamma or by SAC and IFN-gamma, suggesting that these factors may act at the site of inflammation to suppress IL-12 and IFN-gamma production rather than in the lymph node to affect T cell priming. Despite the inability of C5a to inhibit IL-12 production by DCs, the receptor for C5a (CD88) was expressed by these cells, and recombinant C5a induced a Ca2+ flux. Taken together, these results define a range of chemoattractant molecules with the ability to suppress IL-12 production by human monocytes and have broad implications for the regulation of immune responses in vivo.     

Granulocyte/macrophage colony-stimulating factor inhibits IL-12 production of mouse Langerhans cells

We investigated the capacity of mouse Langerhans cells (LC) to produce IL-12, a central cytokine in a Th1 type of immune responses. We prepared purified LC (>95%) from BALB/c mouse skin by the panning method using anti-I-Ad mAb. An ELISA showed that purified LC spontaneously produced IL-12 p40, and that its production was up-regulated following simultaneous stimulation with anti-CD40 mAb and IFN-gamma. Surprisingly, GM-CSF strikingly inhibited IL-12 p40 production by anti-CD40/IFN-gamma-stimulated LC (% inhibition = 97.0 +/- 0.9% at 1 ng/ml GM-CSF). Supernatants of 48-h cultured keratinocytes (KC) also caused the inhibition of LC IL-12 p40 secretion, and this effect was neutralized by anti-GM-CSF mAb. IL-1alpha (1 ng/ml)-stimulated KC produced much more GM-CSF than unstimulated KC (60.9 +/- 0.2 pg/ml vs 20.9 +/- 1.7 pg/ml), and IL-1alpha-stimulated KC supernatants strongly inhibited IL-12 p40 production by anti-CD40/IFN-gamma-stimulated LC (% inhibition = 89.4 +/- 1.4%). A bioassay using an IL-12-dependent T cell line demonstrated the correlation of the level of IL-12 p40 with the bioactivity of IL-12. These results provide important implications for the pathogenesis of atopic dermatitis, which involves the participation of LC and KC with the capacity to produce IL-12 and GM-CSF, respectively.     

Differential effects of LPS and TGF-beta on the production of IL-6 and IL-12 by Langerhans cells, splenic dendritic cells, and macrophages

We examined modulatory effects of lipopolysaccharide (LPS) on IL-6 and IL-12 production by mouse Langerhans cells (LC), spleen-derived CD11c+ dendritic cells (DC), and macrophages (Mphi). Low dose LPS (1 ng/ml) increased IL-6 and IL-12 p40 production by Mphi. LPS slightly augmented IL-6 production but showed no effect on IL-12 p40 production by DC. In contrast, only high dose LPS (1 microg/ml) induced IL-6 but not IL-12 p40 production by LC. CD14 expression was the highest on Mphi and then on DC, but not on LC, which may explain the difference in responsiveness to LPS. We also found that TGF-beta inhibited IL-6 and IL-12 p40 production by LPS-stimulated Mphi. However, TGF-beta did not inhibit IL-6 production and even enhanced IL-12 p40 production by anti-CD40/IFN-gamma-stimulated Mphi. Concerning LC, TGF-beta enhanced IL-6 and IL-12 p40 production when stimulated with anti-CD40/IFN-gamma alone or with anti-CD40/IFN-gamma and LPS. Taken together, these findings indicate diverse effects of LPS and TGF-beta on these antigen presenting cells, which probably represents their differential roles in the innate immunity.     

Altered cytokine release in peripheral blood mononuclear cell cultures from patients with the chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is an idiopathic illness associated with a variety of immunologic abnormalities. To investigate potential pathogenetic mechanisms, we evaluated serum levels and peripheral blood mononuclear cell (PBMC) production of selected cytokines and immunoglobulins. Serum bioactive transforming growth factor beta (TGF-beta) levels were higher (P less than 0.01) in patients with CFS (290 +/- 46 pg/mL) than in control subjects (104 +/- 18 pg/mL), but levels of other cytokines tested were not different. Lipopolysaccharide-stimulated release of interleukin 1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha was increased (P less than 0.05) in PBMC cultures from patients with CFS versus control subjects; enhanced (P less than 0.01) IL-6 release to phytohemagglutinin was also observed. In contrast, TGF-beta release in response to lipopolysaccharide was depressed (P less than 0.01) in PBMC cultures derived from patients with CFS. No differences in IL-2 and IL-4 or immunoglobulin production were observed. The enhanced release of inflammatory cytokines by stimulated PBMC from patients with CFS suggests that these cells are primed for an increased response to immune stimuli. These data also suggest an association between abnormal regulation of TGF-beta production in vivo and in vitro with the immunologic consequence of CFS.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Cytokine-mediated modulation of leptin and adiponectin secretion during in vitro adipogenesis: evidence that tumor necrosis factor-alpha- and interleukin-1beta-treated human preadipocytes are potent leptin producers

Over the last decade, compelling evidence has been presented that cytokines affect adipocyte tissue formation and function. In this study we explored the effect of pro-inflammatory (i.e. interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha) versus anti-inflammatory cytokines (i.e. IL-4, IL-10, and transforming growth factor (TGF)-beta1) on leptin and adiponectin secretion during in vitro human adipogenesis. Confirmative to previous reports, conversion of precursor preadipocytes into mature adipocytes was completely inhibited upon exposure to TNF-alpha, IL-1beta, IFN-gamma, or TGF-beta1. Hence, all these anti-adipogenic cytokines prevented release of adipocyte-specific adiponectin. IFN-gamma also strongly reduced leptin production (> or =85%). However, TNF-alpha, IL-1beta, and TGF-beta1 stimulated leptin production from preadipocytes in the absence of mature adipocytes (20.6+/-5.4 ng/ml, 100.8+/-18.2 ng/ml, and 5.4+/-0.4 ng/ml, respectively, compared to 6.6+/-0.8 ng/ml in control adipocyte cultures on day 21; n=4). IL-4, IL-6 and IL-10 did not, or only slightly, affect adipocyte differentiation and their hormonal secretion. In conclusion, adiponectin and leptin are both synthesized by adipocytes, whereas leptin is also produced by preadipocytes upon TNF-alpha or IL-1beta stimulation. These data suggest that preadipocytes could contribute more to total circulating leptin levels than has been previously considered, especially in diseased conditions were these pro-inflammatory factors play a prominent role.     

In vitro release of interleukin-15 by broncho-alveolar lavage cells and peripheral blood mononuclear cells from patients with different lung diseases

IL-15 shares several biological activities with IL-2 and uses the b and g chain of the IL-2 receptor. In addition to its T-cell stimulating capacity, IL-15 exhibits regulatory properties on macrophage proinflammatory cytokine release. IL-15 is released by non-lymphoid cells, e.g. muscle cells, fibroblasts and monocytes/macrophages. In many lung diseases alveolar macrophages (AM) are activated and release pro- inflammatory cytokines. We asked whether IL-15 is released ex vivo by AM and peripheral blood mononuclear cells (PBMC) from patients with inactive sarcoidosis (PSi), active sarcoidosis (PSa), tuberculosis (TB), hypersensitivity pneumonitis (HSP), cryptogenic fibrosing alveolitis (CFA) and pneumonia (PN). Additionally, we examined the kinetics of the IL-15 release of these cells. During 24 hours of culture, AM from controls (CO) released 3.8 +/- 1.9 pg/ml (mean +/- SD) of IL-15, which was significantly lower than in most of the patient groups (PSa: 8.7 +/- 3.9 pg/ml, TB: 8.4 +/- 1.9 pg/ml, CFA: 5.7 +/- 1.5 pg/ml, and PN: 7. 8 +/- 2.6 pg/ml) except PSi (4.0 +/- 2.6 pg/ml) and HSP (9.3 +/- 9.5 pg/ml). PBMC from patients with PSa released significantly more IL-15 than PBMC from CO (10.8 +/- 8.9 pg/ml versus 6.9 +/- 2.2 pg/ml) whereas PBMC IL-15 release of the other groups did not differ from CO (TB: 5.7 +/- 1.4 pg/ml; CFA: 4.6 +/- 1.6 pg/ml; HSP: 4.9 +/- 3.8 pg/ml). Kinetic studies revealed a minor peak after 5 hours and a major peak from 12 hours to 35 hours for AM and PBMC. In summary, AM from all patient groups but the PSi and the HSP group released increased levels of IL-15, although the total amount of this cytokine is very low.