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The effects of human immunoglobulin preparations for intravenous use (IVIg) on in vitro-induced monokine production were studied. Individual peripheral blood monocytes, obtained from healthy blood donors, which produced interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) after in vitro stimulation, were identified by cytokine-specific monoclonal antibodies (mAb) and indirect immunofluorescence technique. Lipopylosaccharide (LPS) or Borrelia burgdorferi spirochetes (Bb) were used to induce TNF-α and IL-6 production in cultures. Peak synthesis occurred 2.5 hr after initiation of the cultures in tile majority of the monocytes, but not aL all in lymphocytes. The monocytes were identified by two-colour staining using a monocyte-specific mAb. IL-6 was produced by 64 ± 8% or 71 ± 9% (means±SD) of the non-IVIg-exposed monocytes after LPS or Bb stimulation, respectively (n = 12). A dose-dependent and significant reduction of the number of IL-6 producing cells was noted in the IVIg-supplemented cultures (P < 0.003). In these cultures 24 ± 12% or 29 ± 12% of the monocytes made IL-6 in response to LPS or Bb. Kinetic studies indicated a sustained significant inhibition of IL-6 production during 24 hr of culture (P < 0.001). In contrast, TNF-α synthesis was not inhibited by IVIg. LPS or Bb stimulation resulted in 47 ± 18% or 69 ± 7% TNF-α producing cells versus 48 ± 9% or 59 ± 8% in IVIg-supplemented cultures. These results indicate down-regulation of IL-6, but not TNF-α production, by IVIg. A direct antigen neutralization is an unlikely explanation for the divergent effects observed on monokine production after IVIg addition. [ABSTRACT FROM AUTHOR]

Supernatants from rat peritoneal macrophage cultures stimulated with bacterial products contain a Mr 36,000 factor that protects immature cortical thymocytes from loss of viability over a 4-hr incubation period in vitro. This effect could not be produced with purified transforming growth factor-β or recombinant interleukin-6 (IL-6). Further, the partially purified Mr 36,000 fraction was inactive in bioassays for IL-1 and tumour necrosis factor. Maximal production of the factor occurred 2 hr after the addition of 20 µg/ml of lipopolysaccharide, as assessed by the titre resulting in 100% protection of thymocytes in a viability assay. The detection of protective activity within 5 min after addition of the stimulant could be attributed to the release of intracellular stores but protein synthesis was required to account for the increasing titre up to peak levels. The titre fell rapidly after 2 hr so that activity could not be detected at 4 hr. This profile of release was refractory to repeated stimulation with lipopolysaccharide. Conjoint addition of lipopolysaccharide and indomethacin, did, however, allow release in response to subsequent challenge. Related to this finding, prostaglandin E2 completely inhibited the release of protective activity. [ABSTRACT FROM AUTHOR]

Mononuclear cells obtained from human blood were mitogen or antigen activated in vitro in the presence or absence of FK506 or cyclosporin A (CsA). Cytokine production was studied at a single-cell level by ultraviolet (UV) microscopy of fixed permeabilized cells using cytokine-specific monoclonal antibodies (mAb). Phenotypic characterization of the monokine-producing cells was achieved by two-colour immunofluorescent staining. Cytokine production after antigen activation with Staphylococcus aureus enterotoxin A (SEA) was significantly reduced. FK506 or CsA inhibited SEA-induced tumour necrosis factor-alpha (TNF-α) production both in monocytes (P < 0.01) and in lymphocytes (P < 0.01), at a drug concentration of 1-25 ng/ml for FK506 and 100-500 ng/ml for CsA. Lymphocyte synthesis of intcrleukin-2 (IL-2), interferon-gamma (IFN-γ) and TNF-β after SEA activation was also significantly reduced by either of the drugs. In contrast, endotoxin-induced monokine production (TNF-α and IL-6) after lipopolysaccharide (LPS) stimulation was unaffected by FK506 or CsA even when added in concentrations as high as 1000 ng/ml. When the cells were stimulated by phorbol ester (phorbol 12-niyristatc 13-acetatc, PMA) plus calcium ionophore (ionomycin). FK506 and CsA inhibited, in a dose-dependent manner, the production of IL-2, IL-4, IL-5, IFN-γ and TNF-α. The 50% inhibitory concentration (IC50) for FK506 or CsA on the cellular synthesis of the various cytokines varied between 0.6 and 1.0 ng/ml and 20 and 60 ng/ml, respectively. Further stimulation by addition of anti-CD28 mAb to the cultures resulted in an augmented IL-2 and IFN-γ production which was resistant to both FK506 and CsA. This report delineates extensive similarities between the two drugs in mechanisms of immunosuppression by blockade of identical interleukin production. Depending on the mode of cell activation the two drugs inhibited not only cytokine production in lymphocytes but also antigen-induced monokine (TNF-α) production in macrophages, although the optimal immunomodulatory effect of FK506 was achieved at a concentration approximately 50-fold lower than that of CsA. [ABSTRACT FROM AUTHOR]

Treatment with human recombinant turnout necrosis factor-α (rTNFα) significantly enhanced resistance to Listeria monocytogenes infection in mice. The level of protection (which was dose-dependent and maximal at approximately 1·0 μg per mouse) was similar to that previously reported for the monokine rIL-1α, although somewhat greater amounts of rTNFα than rIL-1α were required. Combined administration of suboptimal concentrations of rTNFα and rIL-1α resulted in significant enhancement of resistance beyond that obtained with either monokine alone, whereas further increases in anti-listeria resistance were not observed at doses of rTNFα or IL-1α that were themselves capable of inducing substantial protection. Combined administration of rTNFα and rIL-1α was associated with a delay in onset and lessening in severity of the lymphopenia that accompanied L. monocytogenes infection. The reduced bacterial burden in the spleens and livers of mice treated with rTNFα and rIL-1α was associated with a more rapid decline in serum colony-stimulating activity. Peritoneal macrophages from rTNFα- and rIL-1α-treated listeria-infected mice did not demonstrate enhanced anti-listeria activity in vitro. These results provide further evidence for the potential benefits of rTNFα and other cytokines in promoting anti-bacterial resistance. They further suggest that use of combinations of cytokines is a strategy worthy of further consideration. [ABSTRACT FROM AUTHOR]

CXCR3 chemokines are of particular interest because of their potential involvement in a variety of inflammatory diseases, including the rejection of organ transplants. Although the rat is one of the most appropriate animals for using to study transplantation biology, the structural and functional characteristics of CXCL9 [monokine induced by interferon-γ (Mig)] in this experimental model have not been described. Therefore, we recently conducted a series of experiments to identify and characterize the rat CXCL9 gene. Accordingly, we isolated rat CXCL9 cDNA and genomic DNA. The rat CXCL9 gene encodes a protein of 125 amino acids and spans a 3·5 kbp DNA segment containing four exons in the protein-coding region. We then analysed mRNA expression in various tissues. Transcripts for the gene were found to be expressed at high levels in the lymph nodes and spleen. Then, to confirm the function of the identified gene, rat CXCL9 was transiently expressed in COS-1 cells. Rat recombinant Mig displayed chemotactic properties and induced CXCR3 internalization in CD4+ T cells. Lastly, we analysed the expression of rat CXCL9 in a heterotopic heart allograft model. Both mRNA and protein levels of intragraft CXCL9 were significantly increased following transplantation of ACI to LEW hearts when compared with syngeneic controls. These findings indicate that rat CXCL9 has an in vivo role in the infiltration of CD4+ T cells in the transplanted graft. [ABSTRACT FROM AUTHOR]

Interleukin-10 (IL-10) is a cytokine that regulates various macrophage functions. To elucidate the involvement of endogenous IL-10 in the early stage of murine salmonellosis, we examined the effect of anti-IL-10 monoclonal antibody (mAb) administration on the host defence mechanism against Salmonella choleraesuis infection. The in vivo administration of anti-IL-10 mAb significantly enhanced host resistance at the early stage of Salmonella infection, as assessed by bacterial growth in the peritoneal cavity and the liver. Enhanced levels of monokine mRNA, including IL-1α, tumour necrosis factor-α (TNF-α) and IL-12, were observed from day I after infection in the peritoneal macrophages in anti-IL-10 mAb-treated mice compared with those in control mAb-treated mice. Mice treated with anti-IL-10 mAb exhibited significantly higher levels of interferon-γ (IFN-γ) in the peritoneal exudate's and major histocompatibility complex (MHC) class II expression on the peritoneal macrophages on days 3 and 5 after infection. Notably, in vivo anti-IL-10 mAb brought about an increment of γδ T cells in the peritoneal cavity at the early phase of infection, which was correlated with the expression of endogenous heat-shock protein 60 (HSP60), which is implicated as a potential ligand for γδ T cells, in the infected macrophages. Our results suggest that the neutralization of endogenous IL-10 accelerates some macrophage functions and, consequently, the activation of immunocompetent cells, including & grave;δ cells, at the early stage of infection, resulting in an enhanced host defence against Salmonella infection.Interleukin-10 (IL-10) is a cytokine that regulates various macrophage functions. To elucidate the involvement of endogenous IL-10 in the early stage of murine salmonellosis, we examined the effect of anti-IL-10 monoclonal antibody (mAb) administration on the host defence mechanism against Salmonella choleraesuis infection. The in vivo administration of anti-IL-10 mAb significantly enhanced host resistance at the early stage of Salmonella infection, as assessed by bacterial growth in the peritoneal cavity and the liver. Enhanced levels of monokine mRNA, including IL-1α, tumour necrosis factor-α (TNF-α) and IL-12, were observed from day I after infection in the peritoneal macrophages in anti-IL-10 mAb-treated mice compared with those in control mAb-treated mice. Mice treated with anti-IL-10 mAb exhibited significantly higher levels of interferon-γ (IFN-γ) in the peritoneal exudate's and major histocompatibility complex (MHC) class II expression on the peritoneal macrophages on days 3 and 5 after infection. Notably, in vivo anti-IL-10 mAb brought about an increment of γδ T cells in the peritoneal cavity at the early phase of infection, which was correlated with the expression of endogenous heat-shock protein 60 (HSP60), which is implicated as a potential ligand for γδ T cells, in the infected macrophages. Our results suggest that the neutralization of endogenous IL-10 accelerates some macrophage functions and, consequently, the activation of immunocompetent cells, including & grave;δ cells, at the early stage of infection, resulting in an enhanced host defence against Salmonella infection. [ABSTRACT FROM AUTHOR]

This study was designed to determine if administration of anti-interleukin-4 (IL-4) monoclonal antibody (mAb), interferon-γ, (IFN-γ) and their combination after LP-BM5 retrovirus infection of female C57BL/6 mice would prevent retrovirus-induction of immunosuppression and cytokine dysregulation. Splenic natural killer (NK) cell activity, T- and B-cell proliferation, and T-helper type I (Th1) and Th2 cytokine (IL-2, IFN-γ, IL-5 and IL-10) and monokine [IL-6 and tumour necrosis factor-α (TNF-α)] secretions were monitored, as they are usually altered dramatically after murine retrovirus infection. Administration of IFN-γ, and anti-IL-4 significantly prevented retrovirus-induced suppression of splenic NK cell activity, and splenic T- and B-cell proliferation. They also significantly slowed retrovirus-induced elevation of Th2 cytokine (IL-5 and IL-10) release and monokine (IL-6 and TNF-α) secretion by splenocytes. They prevented the loss of Th 1 cytokine (IL-2 and IFN-γ) release by splenocytes, and alleviated splenomegaly and hypergammaglobulinaemia, precursor signs of development of acquired immune deficiency syndrome (AIDS). These findings could provide insight into the roles of immunomodulator in AIDS treatment as well as the mechanisms by which retrovirus infection induces cytokine dysregulation, facilitating immunodeficiencies in AIDS. [ABSTRACT FROM AUTHOR]

C57Bl/6 female mice fed a Vitamin D (VIT-D)-deficient diet had serum levels of 25-hydroxyvitamin D decreasing with the time of diet exposure (3 and 8 weeks). Cytokine production (IL-6, TNF and IL-1) by peritoneal macrophages cultured in vitro with a standard stimulus, LPS, evaluated in the supernatants as biological activity, was significantly reduced in VIT-D-deficient animals. The defect in monokine production was partial and was evident at suboptimal LPS concentrations and incubation times. I-A antigen expression, induced in macrophages by in vitro exposure to IFN-γ, was not modified in VIT-D-deficient mice, but IFN-γ-inducible macrophage cytotoxicity to tumour target cells was significantly decreased in VIT-D-deficient animals. Moreover, basal and Poly I:C-induced NK activity was not modified by VIT-D deficiency. Thus, macrophage functions, such as cytokine production and tumour cytotoxicity induction, are down-modulated in vitro by VIT-D deprivation. To give more support to the relevance of VIT-D availability for cytokine production, TNF and IL-6 have been evaluated in the sera of control and VIT-D-deficient mice given LPS as a model stimulus. Serum peak levels of both cytokines were at least halved in VIT-D-deprived mice. Thus, VIT-D deficiency may represent a model of partial defect of monokine production. [ABSTRACT FROM AUTHOR]

Macrophage populations derived from Trypanosoma brucei-infected mice suppress both interleukin-2 (IL-2) production and IL-2 receptor expression. To try to identify the regulatory level by which the T-cell activation is switched off, we have analysed the potential of the suppressive macrophage-like cells to block the secretion of the accessory cell-derived T cell co-stimulator interleukin-1 (IL-1). The IL-1 secretion, however, was found to be greatly increased rather than decreased. The increased secretion was in part caused by an increased release rather than by an increased synthesis. In the presence of an in vitro trigger (lipopolysaccharide), the IL-1 secretion was increased 20-30-fold by the infection whereas the total IL-1 production increased only 1.5-2-fold. Macrophages from infected mice thus manifested a marginally increased IL-1 synthesis but released a markedly larger proportion of the synthesized monokine than normal macrophages. In the absence of an in vitro trigger, the infection caused a 10-15-fold increase in IL-1 synthesis as a consequence of an in vivo preactivation. This increase was only observed when the synthesis of prostaglandins was blocked by addition of indomethacin. [ABSTRACT FROM AUTHOR]

During neuronal-induced inflammation, mast cells may respond to stimuli such as neuropeptides in an FcεRI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to immunoglobulin E (IgE)/anti-IgE and compound 48/80. Primary cultured mast cells, generated from CD34+ progenitors in the presence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, concanavalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranulation. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible protein-10, monokine induced by interferon-γ (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte–macrophage colony-stimulating factor, but not IL-4, interferon-γ or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases. [ABSTRACT FROM AUTHOR]

Immunomodulatory dendritic cells (DCs) that induce antigen-specific T-cell tolerance upon in vivo adoptive transfer are promising candidates for immunotherapy of autoimmune diseases. The feasibility of such a strategy has recently proved its efficacy in animal models of allotransplantation and experimental allergic encephalitis, but the effect in inflammatory bowel disease has not yet been demonstrated. In severe combined immunodeficient (SCID) mice, adoptively transferred CD4+ CD25– T cells repopulate the lymphoid tissues and lead to development of chronic colitis characterized by CD4+ T-cell proliferation against enterobacterial extract in vitro. In this model, we adoptively transferred in-vitro-generated bone-marrow-derived DCs exposed to interleukin-10 (IL-10) and an enterobacterial extract. We show that these cells are CD11c positive with intermediate expression of CD40, CD80 and CD86 and have a diminished secretion of IL-6, IL-12 p40/70, tumour necrosis factor-α and keratinocyte-derived chemokine (KC) compared to DCs treated with enterobacterial extract alone. In vivo, these cells prevented weight loss in SCID mice adoptively transferred with CD4+ CD25– T cells, resulted in a lower histopathology colitis score and tended to result in higher serum levels of IL-1α, IL-10, IL-12, IL-13, IL-17, KC and monokine induced by interferon-gamma (MIG). These data underscore the potential of using immunomodulatory DCs to control inflammatory bowel disease and demonstrate its potential use in future human therapeutic settings. [ABSTRACT FROM AUTHOR]

Dendritic cell (DC) subsets play a crucial role in shaping anti‐tumour immunity. Cancer escapes from the control immune system by hijacking DC functions. Yet, bases for such subversion are only partially understood. Tumour cells display aberrant glycan motifs on surface glycoproteins and glycolipids. Such carbohydrate patterns can be sensed by DCs through C‐type lectin receptors (CLRs) that are critical to shape and orientate immune responses. We recently demonstrated that melanoma tumour cells harboured an aberrant 'glyco‐code,' and that circulating and tumour‐infiltrating DCs from melanoma patients displayed major perturbations in their CLR profiles. To decipher whether melanoma, through aberrant glycan patterns, may exploit CLR pathways to mislead DCs and evade immune control, we explored the impact of glycan motifs aberrantly found in melanoma (neoglycoproteins [NeoGP] functionalised with Gal, Man, GalNAc, s‐Tn, fucose [Fuc] and GlcNAc residues) on features of human DC subsets (cDC2s, cDC1s and pDCs). We examined the ability of glycans to bind to purified DCs, and assessed their impact on DC basal properties and functional features using flow cytometry, confocal microscopy and multiplex secreted protein analysis. DC subsets differentially bound and internalised NeoGP depending on the nature of the glycan. Strikingly, Fuc directly remodelled the expression of activation markers and immune checkpoints, as well as the cytokine/chemokine secretion profile of DC subsets. NeoGP interfered with Toll like receptor (TLR)‐signalling and pre‐conditioned DCs to exhibit an altered response to subsequent TLR stimulation, dampening antitumor mediators while triggering pro‐tumoral factors. We further demonstrated that DC subsets can bind NeoGP through CLRs, and identified GalNAc/MGL and s‐Tn/ C‐type lectin‐like receptor 2 (CLEC2) as potential candidates. Moreover, DC dysfunction induced by tumour‐associated carbohydrate molecules may be reversed by interfering with the glycan/CLR axis. These findings revealed the glycan/CLR axis as a promising checkpoint to exploit in order to reshape potent antitumor immunity while impeding immunosuppressive pathways triggered by aberrant tumour glycosylation patterns. This may rescue DCs from tumour hijacking and improve clinical success in cancer patients. [ABSTRACT FROM AUTHOR]

Challenge of the immune system with bacterial superantigens or endotoxin induces the systemic release of cytokines followed by lethal septic shock. The lung is particularly susceptible to systemic toxin exposure resulting in acute leucocyte infiltration and vascular damage. In the present study, the functions of interferon-γ (IFN-γ) and tumour necrosis factor (TNF) for chemokine regulation during acute lung inflammation were examined. Following administration of the superantigen, staphylococcal enterotoxin B (SEB), lung mRNA levels of the chemokines cytokine-induced neutrophil chemo-attractant (KC), lipopolysaccharide-induced CXC chemokine (LIX), macrophage chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α and MIP-2 were increased to a similar extent both in controls and in mice deficient for the IFN-γ or 55 000 MW TNF receptors. In contrast, interferon-inducible protein-10 (IP-10) and monokine induced by IFN-γ (Mig) mRNA expression was markedly reduced in mice deficient for IFN-γ or IFN-γ receptor, but not in 55 000 MW TNF receptor knockout mice. In situ hybridization experiments demonstrated that IP-10 was highly expressed in lung interstitial macrophages of C57BL/6, but not of IFN-γ receptor-deficient mice. In contrast to SEB administration, treatment with lipopolysaccharide resulted in a strong induction of IP-10 and Mig in IFN-γ receptor-deficient mice. Together, these results establish a critical function of IFN-γ for chemokine induction in acute lung inflammation that is dependent on the nature of the inflammatory stimulus. [ABSTRACT FROM AUTHOR]

Polypeptide 48 is a 48 000 MW protein, originally isolated from conditioned media of some human leukaemic cell lines, that induces differentiation and cytolytic activity in HL-60 promyelocytic leukaemia cells and activates human peripheral blood monocytes to secrete interleukin-1 (IL-I) and turnout necrosis factor-α (TNF-α). In the present study we examined the effects of p48 on the accumulation of a series of monokine transcripts, including TNF-α, IL-1α, IL-1β and IL-6, in human peripheral blood monocytes and the myeloid/monocyte cell lines HL-60 and U937. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis, p48 was found to induce accumulation of TNF-α, IL-1α and IL-1β mRNA in peripheral blood monocytes, HL-60 and U937 cells. IL-6 mRNA was found to be increased in p48-stimulated peripheral blood monocytes but not HL-60 or U937. Thus, the secretion of IL-1 and TNF-α by p48-stimulated monocytic cells was associated with up-regulation of cytokine mRNA, suggesting that p48 leads to increased transcription or mRNA stability in these cells. As U937 and HL-60 are likely to represent premonocyte stages of haemopoietic differentiation, it is possible that the effect of p48 on IL-6 mRNA, in contrast to its effect on TNF and IL-1, requires ceils to be at a later differentiation step. [ABSTRACT FROM AUTHOR]

Tumour necrosis factor (TNF) plays a pivotal role in the induction of cerebral complications during Plasmodium falciparum malaria. TNF secretion by macrophages can be induced by lipopolysaccharide (LPS) and by P. falciparum antigens, but it is unclear whether similar mechanisms control the monokine expression in both cases. The signal transduction pathway by which parasite antigens induce TNF secretion remains to be established. The results reported here, using various inhibitors of second messenger pathways, clearly demonstrate that the signal transduction leading to TNF secretion is mediated partly through protein kinase C and calmodulin-dependent protein kinase activation. Furthermore, this signal seems to be differentially regulated after LPS or parasite stimulation, since cyclo-oxygenase inhibition by indomethacin resulted in twofold more TNF production enhancement with LPS stimulation than with parasite stimulation. The nature of the receptor involved in the parasite induced-macrophage stimulation remains obscure. However, the results discussed here indicate that parasite antigens stimulate multiple signal transduction pathways via G protein. Identification of the different pathways involved in these receptor mediated events may be invaluable in the development of specific inhibitors against TNF overproduction during cerebral malaria. [ABSTRACT FROM AUTHOR]

The influence of pooled human IgG preparations for intravenous usc (i.v.Ig) on in vitro-induced cytokine production was studied at the single-cell level using cytokine-specific monoclonal antibodies (mAb) and indirect immunofluorescent technique. Cultured mononuclear cells from peripheral blood from healthy adult donors were polyclonally stimulated for 96 hr by either direct ligation of T-cell receptors using immobilized anti-CD3 mAb or by a combination of a protein kinase C activator [phorbol 12-myristate 13-acetate (PMA)] and a calcium ionophore (ionomycin) in the absence or presence of i.v.Ig. A marked inhibition of proliferation and blast transformation was noted in all i.v.Ig exposed cultures, despite good cell survival. The production of the T-cell lymphokines interleukin-2 (IL-2), IL-10, interferon-γ (IFN-γ) and tumour necrosis factor-β (TNF-β) was significantly down-regulated during the whole studied period in the i.v.Ig containing anti-CD3 stimulated cultures. The synthesis of the monokine IL-8 was not suppressed and that of TNF-α, which was made by both lymphocytes and monocytes, was only moderately inhibited. Somewhat different and more transient effects were observed in the i.v.Ig-exposed PMA/ionomycin-activated cultures. The production of IL-2, IL-3, IL-4, IL-5, IL-10, TNF-β and granulocyte-macrophage colony-stimulating factor (GM-CSF) was down-regulated during the initial phase of the cultures up to 48 hr, but not at 48-96 hr. The synthesis of IFN-γ and TNF-α was unaffected of the influence of i.v.Ig during the entire culture period. The expression of IL-2 receptors (IL-2R) was significantly suppressed in the i.v.Ig-treated anti-CD3-activated cells, but not in the PMA/ionomycin-stimulated cultures. Taken together our results indicate that pooled IgG may mediate immunomodulation by direct effects on cytokine production and on T-cell proliferation. [ABSTRACT FROM AUTHOR]

We have previously described a synergism between the two physiological hormones, retinoic acid (RA) and 1α,25-dihydroxyvitamin D3 (VD) in the induction of U937 cell differentiation towards a more mature state. Herein, we investigated the regulation of cytokine production during RA and/or VD treatment of U937 cells. Cell differentiation was followed by measurement of their capacity to give oxidative responses, and interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and IL-6 gene and protein expression were determined in RA/VD-treated cells, activated or not with lipopolysaccharide (LPS). The undifferentiated and RA-treated U937 cells were unable to produce monokines even when they were stimulated by LPS. VD induced the monokine mRNA expression in U937 cells but failed to induce protein release. However, unlike RA, it primed the cells to secrete monokines upon endotoxin stimulation. A large enhancement of the production of the monokines both at mRNA and protein levels was observed in the U937 cells exposed to the combination of RA + VD. Nevertheless, protein release required a further step of activation of the RA + VD-primed cells. The co-inducer effect of RA and VD was not observed in HL-60 or THP-1 cells and seems to be restricted to U937 cells. These results on cytokine expression support our previous finding that a combination of RA and VD brings the U937 cells to a high stage of myeloid differentiation with major characteristics of monocytes/macrophages. [ABSTRACT FROM AUTHOR]

Leucocytes and vascular cells interact closely in inflammation and immunity and cytokines are important mediators of this interaction. The present study was designed to define the capacity of human endothelial cells (HEC) to produce a monocyte-derived neutrophil chemotactic factor (provisionally termed IL-8). IL-8 is a polypeptide chemotactic for neutrophils originally identified in the culture supernatant of lipopolysaccharide (LPS)-stimulated monocytes. IL-1 induced high levels of production of neutrophil chemotactic activity in culture supernatants of HEC. Optimal stimulation of activity was observed when HEC were cultured with 10–100 ng/ml IL-1β for 16 hr. Anti-IL-8 antibody blocked the chemotactic activity for neutrophils of IL-1-activated HEC supernatants. IL-1-treated HEC expressed high levels of IL-8 mRNA transcripts, as assessed by Northern blot analysis. Tumour necrosis factor (TNF) and LPS, unlike the inflammatory monokine IL-6, also induced IL-8 expression. Nuclear run-off experiments revealed that IL-1 activated transcription of the IL-8 gene. The production of IL-8 may represent a mechanism whereby endothelial cells, exposed to inflammatory signals, participate in the regulation of neutrophil extravasation. [ABSTRACT FROM AUTHOR]

Human synovial fibroblast cell lines (HSN), established from tissues obtained from the knee joints of arthritis patients undergoing arthoplasty, were used to investigate the effects of human interleukin-1 (IL-1) β and turnout necrosis factor (TNF)α on proliferation and prostaglandin E2 (PGE2) secretion. IL-1β aad TNFα were equipotent stimulators of HSN proliferation. Classical non-steroidal anti-inflammatory drugs and glucocorticoids significantly augmented this effect. In addition, IL-1β and TNFα were potent inducers of PGE2 production while exogenous PGE2 was growth inhibiting. These data suggest that the secretion of PGE2 by monokine-stimulated HSN exerts a negative feedback signal. Further examination of IL-1β- and TNFα-induced PGE2 secretion revealed IL-1β to be a more potent stimulator; however, this observation may be due, in part, to differences in the kinetics of induction. Rabbit anti-IL-1β and anti-TNFα specifically neutralized both proliferation and PGE: production induced by these monokines, but anti-IL-1β (or anti-IL-1α) did not block TNFα activity. It is unclear whether TNFα stimulates HSN to produce IL-1, but the antibody data suggest that extracellular IL-1 is not responsible for TNFα in vitro activity. [ABSTRACT FROM AUTHOR]

The injection of BCG vaccine in C57BL/6J mice results in the suppression of the generation of cytotoxic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC) and of mitogenic rections to concanavalin A (Con A). Suppression is mediated by macrophage-like suppressor cells. Since previous work had indicated that suppression involved the inhibition of the production of interlcukin-2 (IL-2), the effects of BCO on interleukin-l (IL-1), a monokine required for IL-2 production, were investigated. It was found that the release of IL-l-like activity in spleen cell cultures stimulated with LPS or Con A was increased by previous BCG treatment of the cell donors. In MLC, the release of IL-l-like activity was also increased by BCG. However, the detection of IL- l -like activity in MLC supernatants was prevented by the presence of a suppressor factor. In this case, the IL-l- like activity could be separated with gel filtration from the suppressor factor which had higher molecular weight. The production of IL-I-like activity by CBA/J spleen cells, which are not suppressed by 8CC, was not significantly different from that of C57BL/63 cells, which are markedly suppressed. Moreover, the addition of IL-I to the BCG-suppressed cultures not only did not restore normal reactivity, but actually further suppressed CTL formation. It was concluded that BCG- induced suppression cannot be attributed to decreased IL-i activity. The suppressor factor discovered during these investigations may have a role in this type of suppression. [ABSTRACT FROM AUTHOR]

The effect of African swine fever virus replication on T and B lymphocytes was studied by mitogen-driven assays. Live attenuated isolates caused a marked suppressive effect which was dose- and time-dependent. This effect appeared to be mediated through a monokine. Live virulent isolates enhanced lymphocyte mitogenesis in most cases, with increased [³H]-thymidine uptake by T cells and increased Ig secretion by B cells. Killed preparation had no effect. These results are discussed in the light of the allergic response and immunopathological lesions produced by African swine fever virus infections. [ABSTRACT FROM AUTHOR]

Local differentiation of eosinophil precursors occurs in the human thymus. Thymic eosinophils are often positioned in the corticomedullary junction between the CD4+CD8+ double‐positive (DP) thymocytes and the CD4+ or CD8+ single‐positive (SP) thymocytes. The aims of this study were to (1) determine if there are distinct thymic eosinophil populations that differ from the blood eosinophil populations and (2) evaluate the capacity of thymic eosinophils to promote the development of SP thymocytes from DP thymocytes. Thymic and blood eosinophils from thymectomized infants (n = 7) were compared regarding the expression of 34 molecules using cytometry by time‐of‐flight (CyTOF). In addition, FACS‐sorted thymic eosinophils were co‐cultured with autologous CD3/CD28‐stimulated DP, CD4 SP, and CD8 SP thymocytes and analysed by flow cytometry and CyTOF. X‐shift clustering analysis and viSNE dimensionality reduction were performed. Seven eosinophil populations were identified within the blood and thymus, respectively, five of which were specific for either tissue. Whereas the blood eosinophil populations varied between individuals, the thymic eosinophil populations were more uniform. The eosinophil‐thymocyte co‐cultures resulted in (1) an increase in CD4 SP thymocytes when eosinophils were cultured with DP thymocytes, (2) decreased frequency of CD8 SP thymocytes when these were cultured with eosinophils, and (3) a more mature thymic phenotype when eosinophils were cultured with CD4 SP thymocytes. Thymic eosinophils are a specialized population of eosinophils with a distinct phenotype that separates them from their blood counterparts, and in vitro they appear to favour CD4 SP thymocyte development to the detriment of CD8 SP thymocytes. [ABSTRACT FROM AUTHOR]

Chronic lung allograft dysfunction (CLAD) remains the major barrier to long‐term survival after lung transplantation and improved insight into its underlying immunological mechanisms is critical to better understand the disease and to identify treatment targets. We systematically searched the electronic databases of PubMed and EMBASE for original research publications, published between January 2000 and April 2021, to comprehensively assess current evidence on effector immune cells in lung tissue and bronchoalveolar lavage fluid from lung transplant recipients with CLAD. Literature search revealed 1351 articles, 76 of which met the criteria for inclusion in our analysis. Our results illustrate significant complexity in both innate and adaptive immune cell responses in CLAD, along with presence of numerous immune cell products, including cytokines, chemokines and proteases associated with tissue remodelling. A clear link between neutrophils and eosinophils and CLAD incidence has been seen, in which eosinophils more specifically predisposed to restrictive allograft syndrome. The presence of cytotoxic and T‐helper cells in CLAD pathogenesis is well‐documented, although it is challenging to draw conclusions about their role in tissue processes from predominantly bronchoalveolar lavage data. In restrictive allograft syndrome, a more prominent humoral immune involvement with increased B cells, immunoglobulins and complement deposition is seen. Our evaluation of published studies over the last 20 years summarizes the complex multifactorial immunopathology of CLAD onset and progression. It highlights the phenotype of several key effector immune cells involved in CLAD pathogenesis, as well as the paucity of single cell resolution spatial studies in lung tissue from patients with CLAD. [ABSTRACT FROM AUTHOR]

CXCR3, predominately expressed on memory/activated T cells, is a receptor for both interferon-γ inducible protein-10/CXC ligand 10 (CXCL10) and monokine induced by interferon-γ/CXCL9. We reported here that CXCR3 was highly up-regulated on infiltrating eosinophils in Schistosoma japonicum egg-induced granuloma in the mouse liver. It was also highly and functionally up-regulated on peritoneal exudate eosinophils in mice infected with S. japonicum. The phenomena were demonstrated at protein and mRNA levels using immunohisto- and immunocytochemistry evaluation of biopsy, flow cytometry and real-time quantitative reverse transcriptase–polymerase chain reaction technique, and verified by Northern blotting and chemotaxis assay in vitro. We also found that CCR3 expression on the infiltrating and peritoneal exudate cells was significantly decreased, CXCR4 expression was unchanged during the 42-day period of infection. We screened mRNA expression levels of the all known chemokine receptors in purified peritoneal exudate eosinophils and liver granuloma dominated by eosinophils. CXCR3 was highly and functionally up-regulated on peritoneal exudate eosinophils in mice infected with S. japonicum, meanwhile CCR3 was significantly and functionally down-regulated in these cells. The findings could lead to a better understanding of the chemokine receptor expression pattern of eosinophils at inflamed tissue sites caused by parasites. These could be also crucial for establishing a therapeutic strategy for eosinophilic inflammation via intervention in chemokine actions. [ABSTRACT FROM AUTHOR]

Causes of individual variation in susceptibility to mycobacterial diseases are only partly understood. An efficient cell-mediated immune response is crucial for resistance. Macrophages and T cells interact to eliminate the mycobacteria, partially through the effects of secreted cytokines. A vigorous anti-bacterial inflammatory response is sometimes accompanied by severe tissue damage, while immunosuppression leads to progressive infection. Here, live, attenuated Mycobacterium bovic, bacillus Calmette-Guérin (BCG), was used as a model antigen to study cytokine production at the single-cell level in response to mycobacteria. Peripheral blood mononuclear cells from healthy individuals were challenged in vitro and the kinetics and frequencies of cytokine-producing cells were studied by immunofluorescent visualization of intracellular cytokines. Fourteen cytokines were assayed; interleukin-1α (IL-1α), IL-1β, IL-I receptor antagonist (IL-1ra), IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), TNF-β and granulocyte-macrophage colony-stimulating factor (GM-CSF). A sequential production of T helper-1 (Th 1) and T helper-2 (Th2) cytokines was induced by BCG. Early, at days 1-2 after stimulation, the response was dominated by monokines and a low IFN-γ and TNF-β production. At days 4-5 there was a marked production of Th1 lymphokines, with approximately 6% IFN-γ+ cells, 4% TNF-β+ cells and 2% IL-2+ cells. Late in the reaction, at days 10-12, a Th2 response with IL-4, IL-5 and IL-10 was detected, while the synthesis of Th1 lymphokines and monokines declined. Overall, our results provide further evidence of IFN-γ as the major cytokine induced by mycobacteria in healthy individuals, but also suggest that Th2 cytokines participate in the response. [ABSTRACT FROM AUTHOR]

Zinc is known to be greatly involved in the regulation of immune functions. Pharmacological zinc supplementation, leading to serum zinc concentrations of more than 0.025 mM, has often been suggested to improve immune responses. However, the exact influence of elevated zinc level on immune functions has not yet been investigated. We found that zinc level selectively enhances cytokine Suction by lipopolysaccharidc (LPS) in a concentration-dependent fashion: as little as 0.0125 mM supplemental zinc led to nearly 50% elevated interleukin-1β (IL-1β) levels both in polymorphonuclear cells (PBMC) and whole-blood cultures. The secretion of interferon-γ (IFN-γ) could be increased more than 10-fold by 0.1 mM zinc. This could not be observed during stimulation with phytohaemagglutin (PHA). In contrast, zinc levels concentration-dependently down-regulated monocyte activation caused by the superantigens, staphylococcal enterotoxins A and E (SEA, SEE, more than 90% down-regulation by 0.1 mM zinc), the Mycoplasma anthritidis-derived superantigen (MAS), but not toxic shock syndrome toxin-1 (TSST-1), while T-cell response remained unaffected. This was not the result of chemical degradation of the superantigens. We assume that zinc concentration regulates interactions between SEA, SEE and MAS, but not TSST- 1 and their major histocompatibility complex (MHC) class II-binding sites. Our data demonstrate that zinc levels control the secretion of IFN-γ and monokines after both LPS and superantigen challenge within a clinically relevant range of concentrations. This reveals new perspectives and indications for zinc supplementation and also indicates potential risks of therapeutic application of zinc. [ABSTRACT FROM AUTHOR]

Incubation of human neutrophils with recombinant human interleukin-1α (IL-1α) resulted in the suppression of neutrophil adherence. In contrast, similar treatment with recombinant human tumour necrosis factor-α (TNFα, cachectin) resulted in the enhancement of neutrophil adherence. These contrasting effects were noted as early as 5 min after incubation, and persisted for at least 60 min. Simultaneous addition of these two monokines resulted in intermediate values between suppression by IL-1 and enhancement by TNFα. The stimulatory effects of the chemotactic peptide FMLP and the phorbol ester PMA were ameliorated by IL-1 but augmented by TNFα. The effects of these monokines on neutrophil adherence were abolished by heating but not by polymyxin B treatment, showing that their modulatory properties were not mediated by endotoxin. [ABSTRACT FROM AUTHOR]

The influence of human monocyte-derived chemotactic peptide (GCP/IL-8) on degranulation of neutrophils was investigated in relation to that of other monokines. GCP/IL-8 promoted a dose- dependent release of lactoferrin from specific granules but had no effect on enzyme release from primary granules. From the other monokines that were tested, tumour necrosis factor alpha (TNFα) also induced degranulation, while IL-1β and IL-6 scored negatively. TNFα-induced lactoferrin release was enhanced by cytochalasin B pretreatment of the granulocytes, while GCP/IL-8-promoted degranulation was not. In contrast to GCP, TNFα also caused the release of LDH, suggesting a non- specific cell destruction. These observations further support the view that, unlike the other monokines, GCP/ IL-8 is a true and specific granulocyte activator. [ABSTRACT FROM AUTHOR]

Summary: Regulatory T (Treg) cells play an essential role in the maintenance of intestinal homeostasis. In Peyer's patches (PPs), which comprise the most important IgA induction site in the gut‐associated lymphoid tissue, Treg cells promote IgA isotype switching. However, the mechanisms underlying their entry into PPs and isotype switching facilitation in activated B cells remain unknown. This study, based on the dextran sulphate sodium (DSS)‐induced colitis model, revealed that Treg cells are significantly increased in PPs, along with CD11b+ B‐cell induction. Immunofluorescence staining showed that infiltrated Treg cells were located around CD11b+ B cells and produced transforming growth factor‐β, thereby inducing IgA+ B cells. Furthermore, in vivo and in vitro studies revealed that CD11b+ B cells in PPs had the capacity to recruit Treg cells into PPs rather than promoting their proliferation. Finally, we found that Treg cell recruitment was mediated by the chemokine CXCL9 derived from CD11b+ B cells in PPs. These findings demonstrate that CD11b+ B cells induced in PPs during colitis actively recruit Treg cells to accomplish IgA isotype switch in a CXCL9‐dependent manner. This study provides evidence that CD11b+ B cells are induced in PPs, in a DSS‐induced colitis model. On the course of the disease, induced CD11b+ B cells can attract Treg cells into PPs in a CXCL9‐dependent manner. [ABSTRACT FROM AUTHOR]

The success of immune system-based cancer therapies depends on a broad immune response engaging a range of effector cells and mechanisms. Immune mobilizing monoclonal T cell receptors ( TCRs) against cancer (Imm TAC™ molecules: fusion proteins consisting of a soluble, affinity enhanced TCR and an anti- CD3 scFv antibody) were previously shown to redirect CD8+ and CD4+ T cells against tumours. Here we present evidence that IMCgp100 (Imm TAC recognizing a peptide derived from the melanoma-specific protein, gp100, presented by HLA-A*0201) efficiently redirects and activates effector and memory cells from both CD8+ and CD4+ repertoires. Using isolated subpopulations of T cells, we find that both terminally differentiated and effector memory CD8+ T cells redirected by IMCgp100 are potent killers of melanoma cells. Furthermore, CD4+ effector memory T cells elicit potent cytotoxic activity leading to melanoma cell killing upon redirection by IMCgp100. The majority of T cell subsets belonging to both the CD8+ and CD4+ repertoires secrete key pro-inflammatory cytokines (tumour necrosis factor- α, interferon- γ, interleukin-6) and chemokines (macrophage inflammatory protein-1 α- β, interferon- γ-inducible protein-10, monocyte chemoattractant protein-1). At an individual cell level, IMCgp100-redirected T cells display a polyfunctional phenotype, which is a hallmark of a potent anti-cancer response. This study demonstrates that IMCgp100 induces broad immune responses that extend beyond the induction of CD8+ T cell-mediated cytotoxicity. These findings are of particular importance because IMCgp100 is currently undergoing clinical trials as a single agent or in combination with check point inhibitors for patients with malignant melanoma. [ABSTRACT FROM AUTHOR]

Murine contact sensitivity ( CS) reaction could be antigen-specifically regulated by T CD8+ suppressor (Ts) lymphocytes releasing micro RNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (M φ). The present studies investigated the role of M φ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of M φ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of M φ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of M φ, demonstrating the substantial role of M φ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl ( TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed M φ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated M φ. Additionally, exosome-pulsed, TNP-conjugated M φ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated M φ in a transmembrane manner was observed. Our results demonstrated the essential role of M φ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation. [ABSTRACT FROM AUTHOR]

Keratinocyte-associated C-type lectin ( KACL) is a peculiar C-type lectin-like receptor ( CTLR) due to its selective expression by human keratinocytes and cognate interaction with the genetically coupled CTLR NKp65. KACL and NKp65 are members of the CLEC2 and NKRP1 subfamilies of natural killer gene complex ( NKC)-encoded CTLR, respectively. Most NKRP1 molecules are expressed on NK cells and T cells and act as receptors of CLEC2 glycoproteins with their genes being intermingled in a certain sub-region of the mammalian NKC. The reasons for the tight genetic linkage of these dedicated receptor/ligand pairs are unknown, as is the physiological expression of NKp65. Recently, we reported that the CTLR NKp65 and KACL interact with high affinity, resulting in activation of NKp65-expressing NK-92 MI cells. Here, we address the molecular basis of this high-affinity interaction by analysing KACL mutants with KACL-specific monoclonal antibodies ( mAb), soluble NKp65 ( sNKp65) and NK-92 MI- NKp65 cells. We find that none of the three N-linked carbohydrates of KACL glycoproteins significantly contributes to KACL surface expression and NKp65 interaction. However, KACL mutants with non-conservative amino acid substitutions of arginine 158 or isoleucine 161 abrogated binding of both KACL-specific mAb OMA1 and sNKp65, well in line with the blockade of NKp65- KACL interaction by OMA1. Accordingly, functional recognition of these KACL mutants by NK-92M- NKp65 cells was completely abolished. Arginine 158 and isoleucine 161 located at the membrane-distal surface of KACL were defined as residues, decisively determining functional KACL- NKp65 interaction that is independent of KACL glycosylation. [ABSTRACT FROM AUTHOR]

Human cathelicidin LL-37 protects against infections and endotoxin-induced inflammation. In a recent study we have shown that IG-19, an LL-37-derived peptide, protects in a murine model of arthritis. Cytokine interleukin-32 ( IL-32) is elevated and directly associated with the disease severity of inflammatory arthritis. Therefore, in this study we examined the effects of LL-37 and IG-19 on IL-32-induced responses in human peripheral blood-derived mononuclear cells ( PBMC) and macrophages. We showed that CD14+ monocytes are the primary cells that produce pro-inflammatory tumour necrosis factor- α ( TNF- α) following stimulation of PBMC with IL-32. We demonstrated that LL-37 and IG-19 significantly suppress IL-32-induced production of pro-inflammatory cytokines, e.g. TNF- α and IL-1 β, without altering chemokine production. In contrast, LL-37 and IG-19 enhance the production of the anti-inflammatory cytokine IL-1 RA. Further mechanistic studies revealed that LL-37 and IG-19 suppress IL-32-mediated phosphorylation of Fyn (Y420) Src kinase. In contrast, IL-32-mediated phosphorylation of AKT-1 (T308) and MKP-1 (S359) is not suppressed by the peptides. LL-37 and IG-19 alone induce the phosphorylation of MKP-1 (S359), which is a known negative regulator of inflammation. Furthermore, the peptides induce the activity of p44/42 mitogen-activated protein kinase, which is known to phosphorylate MKP-1 (S359). This is the first study to demonstrate the regulation of IL-32-induced inflammation by LL-37 and its derivative peptide IG-19. The mechanistic results from this study suggest that regulation of immune-mediated inflammation by these peptides may be controlled by the dual phosphatase MKP-1. We speculate that LL-37 and its derivatives may contribute to the control of immune-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

Acquisition of non-sterilizing natural immunity to Plasmodium falciparum malaria has been shown in low transmission areas following multiple exposures. However, conflicting data from endemic areas suggest that the parasite may interfere with the induction of effective B-cell responses. To date, the impact of blood-stage parasite exposure on antigen-specific B cells has not been reported following controlled human malaria infection ( CHMI). Here we analysed human B-cell responses in a series of Phase I/IIa clinical trials, which include CHMI, using candidate virus-vectored vaccines encoding two blood-stage antigens: merozoite surface protein 1 ( MSP1) and apical membrane antigen 1 ( AMA1). Previously vaccinated volunteers show boosting of pre-existing antigen-specific memory B-cell (m BC) responses following CHMI. In contrast, unvaccinated malaria-naive control volunteers developed an m BC response against MSP1 but not AMA1. Serum Ig G correlated with the m BC response after booster vaccination but this relationship was less well maintained following CHMI. A significant reduction in peripheral MSP1-specific m BC was observed at the point of diagnosis of blood-stage infection. This was coincident with a reduction in peripheral blood B-cell subsets expressing CXCR3 and elevated serum levels of interferon- γ and CXCL9, suggesting migration away from the periphery. These CHMI data confirm that m BC and antibody responses can be induced and boosted by blood-stage parasite exposure, in support of epidemiological studies on low-level parasite exposure. [ABSTRACT FROM AUTHOR]

Human β defensin-3 (h BD-3) is an antimicrobial peptide with diverse functionality. We investigated the capacity of hBD-3 and, for comparison, Pam3 CSK4 and LL-37 to induce co-stimulatory molecules and chemokine expression in monocytes. These stimuli differentially induced CD80 and CD86 on the surface of monocytes and each stimulant induced a variety of chemokines including monocyte chemoattractant protein 1 ( MCP-1), Gro-α, macrophage-derived chemokine ( MDC) and macrophage inflammatory protein 1β ( MIP1β), while only h BD-3 and Pam3 CSK4 significantly induced the angiogenesis factor, vascular endothelial growth factor (VEGF). Human BD-3 induced similar chemokines in monocyte-derived macrophages and additionally induced expression of Regulated upon activation normal T-cell expressed and presumably secreted ( RANTES) in these cells. Comparison of monocytes from HIV+ and HIV- donors indicated that monocytes from HIV+ donors were more likely to spontaneously express certain chemokines (MIP-1α, MIP-1β and MCP-1) and less able to increase expression of other molecules in response to hBD-3 (MDC, Gro-α and VEGF). Chemokine receptor expression ( CCR5, CCR2 and CXCR2) was relatively normal in monocytes from HIV+ donors compared with cells from HIV- donors with the exception of diminished expression of the receptor for MDC, CCR4, which was reduced in the patrolling monocyte subset ( CD14+ CD16++) of HIV+ donors. These observations implicate chemokine induction by h BD-3 as a potentially important mechanism for orchestrating cell migration into inflamed tissues. Alterations in chemokine production or their receptors in monocytes of HIV-infected persons could influence cell migration and modify the effects of h BD-3 at sites of inflammation. [ABSTRACT FROM AUTHOR]

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists. [ABSTRACT FROM AUTHOR]

Vaccine adjuvants activate the innate immune system and thus influence subsequent adaptive T-cell responses. However, little is known about the initial immune mechanisms preceding the adjuvant-induced differentiation of T-helper (Th) cells. The effect of a T-helper 1 (Th1) adjuvant, dimethyldioctadecylammonium liposomes with monophosphoryl lipid-A (DDA/MPL), and a T-helper 2 adjuvant, aluminium hydroxide [Al(OH)3], on early, innate chemotactic signals and inflammatory cell influx at the site of injection was therefore investigated. Injection of the adjuvants into the peritoneal cavity of mice demonstrated distinct differences in the magnitude, quality and kinetics of the response. The inflammatory response to DDA/MPL was prominent, inducing high local levels of pro-inflammatory cytokines, chemokines and a pronounced inflammatory exudate consisting of neutrophils, monocytes/macrophages and activated natural killer cells. This was in contrast to the response induced by Al(OH)3, which, although sharing some of the early chemokine signals, was more moderate and consisted almost exclusively of neutrophils and eosinophils. Notably, Al(OH)3 specifically induced the release of a significant amount of interleukin (IL)-5, whereas DDA/MPL induced high amounts of tumour necrosis factor-α (TNF-α), IL-1α and IL-6. Finally, a microarray analysis confirmed that the effect of DDA/MPL was broader with more than five times as many genes being specifically up-regulated after injection of DDA/MPL compared with Al(OH)3. Thus, the adjuvants induced qualitatively distinct local inflammatory signals early after injection. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte–macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam3Cys-Ser-(Lys)4 (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses. [ABSTRACT FROM AUTHOR]

The mammalian immune system is intricately regulated, allowing for potent pathogen-specific immunity to be rapidly activated in response to infection with a broad and diverse array of potential pathogens. As a result of their ability to differentiate into distinct effector lineages, CD4 T cells significantly contribute to pathogen-specific adaptive immune responses. Through the production of effector cytokines, CD4 T helper (Th) cells orchestrate the precise mobilization of specific immune cells to eradicate infection. The protective effects of the newly identified lineage of Th17 cells against pathogens like Klebsiella pneumoniae, Citrobacter rodentium and Candida albicans indicate the capacity of Th17 cells to confer protection against extracellular bacterial and fungal pathogens, filling a critical void in host immunity not covered by the classically described Th1 lineage that activates immunity to intracellular pathogens or the Th2 lineage that is important in protection against mucosal parasitic pathogens. Host defence by Th17 cells extends beyond protection against extracellular bacterial and fungal pathogens, as demonstrated in infections against intracellular bacteria like Listeria monocytogenes and Salmonella enterica, as well as Mycobacterium tuberculosis. Herein, we summarize both experimental data from mouse infection models and epidemiological studies in humans that demonstrate the protective effects of interleukin-17 and Th17 CD4 T cells in immunity to bacterial, mycobacterial and fungal pathogens. [ABSTRACT FROM AUTHOR]

Man has moved rapidly from the hunter–gatherer environment to the living conditions of the rich industrialized countries. The hygiene hypothesis suggests that the resulting changed and reduced pattern of exposure to microorganisms has led to disordered regulation of the immune system, and hence to increases in certain inflammatory disorders. The concept began with the allergic disorders, but there are now good reasons for extending it to autoimmunity, inflammatory bowel disease, neuroinflammatory disorders, atherosclerosis, depression associated with raised inflammatory cytokines, and some cancers. This review discusses these possibilities in the context of Darwinian medicine, which uses knowledge of evolution to cast light on human diseases. The Darwinian approach enables one to correctly identify some of the organisms that are important for the ‘Hygiene’ or ‘Old Friends’ hypothesis, and to point to the potential exploitation of these organisms or their components in novel types of prophylaxis with applications in several branches of medicine. [ABSTRACT FROM AUTHOR]

Although intraocular tumours reside in an immune-privileged site, they can circumvent immune privilege and undergo rejection. Ocular tumour rejection typically follows one of two pathways. One pathway involves CD4+ T cells, delayed-type hypersensitivity (DTH), and culmination in ischemic necrosis of the tumour and phthisis (atrophy) of the eye. The second pathway is DTH-independent and does not inflict collateral injury to ocular tissues, and the eye is preserved. In this study, we used a well-characterized tumour, Ad5E1, to investigate the role of CD4+ T cells in the non-phthisical form of intraocular tumour rejection. It has been previously documented that CD4+ T cells and interferon (IFN)-γ are necessary for rejection of these tumours in the eye. In this study, we demonstrate that CD4+ T cells can circumvent immune privilege and infiltrate intraocular Ad5E1 tumours. Following tumour rejection, CD4+ T cells from tumour rejector mice could be adoptively transferred to severe combined immunodeficiency (SCID) mice and protect them from intraocular Ad5E1 tumour growth. Tumour-specific CD4+ T cells produced IFN-γ in response to Ad5E1 tumour antigens. Macrophages also contributed to rejection, as they were present in intraocular Ad5E1 tumours, and local depletion of macrophages resulted in progressive tumour growth. Ocular macrophages contributed to Ad5E1 tumour rejection, as Ad5E1 tumour rejection did not occur in macrophage-depleted SCID mice reconstituted with rejector CD4+ T cells. This demonstrates that macrophage and CD4+ T-cell co-operation is needed for non-phthisical rejection of intraocular tumours. [ABSTRACT FROM AUTHOR]

Heterotrimeric Gi proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS-induced, heat-killed SA-induced and heat-killed GBS-induced cytokine and chemokine production in peritoneal macrophages from wild-type (WT), Gαi2–/– or Gαi1/3–/– mice were investigated. LPS induced production of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10 and interferon-γ-inducible protein-10 (IP-10); SA induced TNF-α, and IL-1β production; and GBS induced TNF-α, IL-6, IL-1β, macrophage inflammatory protein-1α (MIP-1α) and keratinocyte chemoattract (KC) production were all decreased ( P < 0·05) in Gαi2–/– or Gαi1/3–/– mice compared with WT mice. In contrast to the role of Gi proteins as a positive regulator of mediators, LPS-induced production of MIP-1α and granulocyte–macrophage colony-stimulating factor (GM-CSF) were increased in macrophages from Gαi1/3–/– mice, and SA-induced MIP-1α production was increased in both groups of Gαi protein-depleted mice. LPS-induced production of KC and IL-1β, SA-induced production of GM-CSF, KC and IP-10, and GBS-induced production of IL-10, GM-CSF and IP-10 were unchanged in macrophages from Gαi2–/– or Gαi1/3–/– mice compared with WT mice. These data suggest that Gi2 and Gi1/3 proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram-positive microbial stimuli. [ABSTRACT FROM AUTHOR]

The chemokine receptor CXCR3 is critical for the function of activated T cells. We studied the molecular mechanisms of CXCR3 signalling. The addition of CXCR3 ligands to normal human T cells expressing CXCR3 led to the tyrosine phosphorylation of multiple proteins. Addition of the same ligands to Jurkat T cells engineered to express CXCR3 induced tyrosine phosphorylation of proteins with molecular weights similar to those in normal cells. Immunoblotting with phosphotyrosine-specific antibodies identified Zeta-associated protein of 70 000 molecular weight (ZAP-70), linker for the activation of T cells (LAT), and phospholipase-C-γ1 (PLCγ1) to be among the proteins that become phosphorylated upon CXCR3 activation. ZAP-70 was phosphorylated on tyrosine 319, LAT on tyrosines 171 and 191, and PLCγ1 on tyrosine 783. The ZAP-70 inhibitor piceatannol reduced CXCR3-mediated tyrosine phosphorylation of ZAP-70, LAT, PLCγ1 and mitogen-activated protein kinase Erk and it reduced CXCL10-mediated chemotaxis of both CXCR3-transfected Jurkat T cells and normal T cells expressing CXCR3. These results are consistent with the involvement of ZAP-70 in CXCR3-mediated protein tyrosine phosphorylation and CXCR3-induced T-cell chemotaxis. Studies with the Lck-deficient Jurkat T-cell line, JCAM1.6, demonstrated that phosphorylation of ZAP-70 after CXCR3 activation is a Lck-dependent process. Finally, stimulating CXCR3-expressing Jurkat T cells and normal T cells expressing CXCR3 through the T-cell receptor attenuated CXCR3-induced tyrosine phosphorylation and CXCR3-mediated T-cell migration, indicating the occurrence of cross-talk between T-cell receptor and CXCR3-signalling pathways. These results shed light on the mechanisms of CXCR3 signalling. Such information could be useful when designing therapeutic strategies to regulate T-cell function. [ABSTRACT FROM AUTHOR]

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4+ T helper 1 (Th1) cells, while recovery from the disease is associated with the presence of Th2 cells. Here we used animals with targeted deletion of the T-bet gene to determine its role in the progression of EAE. T-bet regulates the production of interferon-γ (IFN-γ) in CD4+ and natural killer cells, and CD4+ T cells from T-bet-deficient mice were unable to differentiate into a Th1 phenotype. Moreover BALB/c mice deficient in T-bet were resistant to the induction of EAE disease, with minimal inflammatory infiltrates in the central nervous system. These mice were resistant to EAE induction even when PLP(180−199) peptide specific effector T cells from BALB/c wild type were transferred to BALB/c T-bet-deficient mice. This resistance to EAE is may be caused by the production of the anti-inflammatory cytokine interleukin-10 (IL-10) from the spleen cells upon ex vivo stimulation with PLP(180−199) peptide and in vivo presence in the central nervous system. There was no difference in the recall responses in spleen cells from T-bet-deficient and wild type mice; however, less secretion of IFN-γ was observed from primed splenocytes. The expression of IFN-γ was less in the central nervous system of T-bet-deficient mice whereas IL-10 was significantly higher in T-bet-deficient as compared to wild type mice. These data indicate that T-bet genes play a critical role in maintaining the encephalitogenic nature of CD4+ T cells in autoimmune responses during EAE disease progression. [ABSTRACT FROM AUTHOR]

Diverse immunotherapy approaches have achieved success in controlling individual aspects of immune responses in animal models. Transfer of such immunotherapies to clinical trials has obtained some success in patients, with clinical responses observed or effective antigen specific immune responses achieved, but has had limited impact on patient survival. Key elements required to generatede novocell-mediated antitumour immune responsesin vivoinclude recruitment of antigen-presenting cells to the tumour site, loading these cells with antigen, and their migration and maturation to full antigen-presenting function. In addition, it is essential for antigen-specific T cells to locate the tumour to mediate cytotoxicity, emphasizing the need for local inflammation to target effector cell recruitment. We review those therapies that involve the tumour site as a target and source of antigen for the initiation of immune responses, and discuss strategies to generate and co-ordinate an optimal cell-mediated immune response to control tumours locally. [ABSTRACT FROM AUTHOR]

Dendritic cells (DC) play critical roles in generating an immune response and in inducing tolerance. Diverse microenvironmental factors can‘polarize’ DC toward an immunogenic or non-immunogenic phenotype. Among the various microenvironmental factors, interleukin-10 (IL-10) exhibits a potent immunosuppressive effect on antigen-presenting cells (APC). Here, we show that monocyte-derived DC generated in the presence of IL-10 exhibit a profound down-regulation of many genes that are associated with immune activation and show that the IL-10-grown DC are poor stimulators of CD8+ T cells in a strictly autologous and major histocompatibility complex (MHC) class I-restricted melanoma antigen recognized by T cells (MART-1)epitope presentation system. However, these IL-10-grown DC can efficiently activate the epitope-specific CD8+ T cells when they are made to present the epitope following transduction with an adenoviral vector expressing the MART-1 antigen. In addition, we show that the MART-1 protein colocalizes with the MHC class I protein, equally well, in the iDC and in the DC cultured in presence of IL-10 when both DC types are infected with the viral vector. We also show that the vector transduced DC present the MART-127−35 epitope for a sustained period compared to the peptide pulsed DC. These data suggest that although DCs generated in the presence of IL-10 tend to be non-immunogenic, they are capable of processing and presenting an antigen when the antigen is synthesized within the DC. [ABSTRACT FROM AUTHOR]

As current research illuminates the dynamic interplay between the innate and acquired immune responses, the interaction and communication between these two arms has yet to be fully investigated. Polymorphonuclear neutrophils (PMNs) and interferon-γ (IFN-γ) are known critical components of innate and acquired immunity, respectively. However, recent studies have demonstrated that these two components are not entirely isolated. Treatment of PMNs with IFN-γ elicits a variety of responses depending on stimuli and environmental conditions. These responses include increased oxidative burst, differential gene expression, and induction of antigen presentation. Many of these functions have been overlooked in PMNs, which have long been classified as terminal phagocytic cells incapable of protein synthesis. As this review reports, the old definition of the PMN is in need of an update, as these cells have demonstrated their ability to mediate the transition between the innate and acquired immune responses. [ABSTRACT FROM AUTHOR]

Granuloma formation in response to mycobacterial infections is associated with increased expression of inducible nitric oxide synthase (NOS2) within granuloma macrophages and increased levels of nitrate/nitrite in the sera of infected mice. Continuous treatment with 5 mm or 10 mm l-N[sup 6]-(1-imino-ethyl)-lysine (L-NIL), a selective NOS2-inhibitor, in acidified drinking water for up to 7 weeks consistently reduced infection-induced nitrate/nitrite to background levels in mycobacteria-infected BALB/c mice. Oral treatment with 5 mm L-NIL initiated at the time of infection significantly exacerbated growth of Mycobacterium tuberculosis, but had no effect on Mycobacterium avium colony-forming unit development in the liver, spleen and lungs of intravenously infected mice. In order to examine the role of nitric oxide in mycobacteria-induced granulomatous inflammation in the absence of any effect on the bacterial load, M. avium-infected mice were treated with 5 mm L-NIL from day 1 through 38 and the development of granulomatous lesions in the liver was assessed by histology, immunohistology and reverse-transcription–polymerase chain reaction (RT-PCR). Computer- and video-assisted morphometry performed at 4 and 7 weeks post-infection showed that treatment with L-NIL led to markedly increased number, cellularity and size of granulomatous lesions in infected mice regardless of the virulence of the M. avium isolate used for infection. Immunohistology of the liver revealed that in mice treated with L-NIL, the numbers of CD3[sup +] T cells, CD21/35[sup +] B cells, CD11b[sup +] macrophages and RB6-8C5[sup +] granulocytes associated with granulomatous lesions was increased. RT-PCR of the liver showed that in L-NIL-treated mice infected with M. avium, mRNA levels of tumour necrosis factor, interleukin-12p40, interferon-γ, interleukin-10 and interferon-γ-inducible protein-10 (IP-10) were up-regulated, while mRNA levels of interleukin-4, monocyte chemotacti... [ABSTRACT FROM AUTHOR]

While bacterial DNA and cytosine–guanosine-dinucleotide-containing oligonucleotides (CpG ODN) are well described activators of murine immune cells, their effect on human cells is inconclusive. We investigated their properties on human peripheral blood mononuclear cells (PBMC) and subsets thereof, such as purified monocytes, T and B cells. Here we demonstrate that bacterial DNA and CpG ODN induce proliferation of B cells, while other subpopulations, such as monocytes and T cells, did not proliferate. PBMC mixed cell cultures, as well as purified monocytes, produced interleukin-6 (IL-6), IL-12 and tumour necrosis factor-α upon stimulation with bacterial DNA; however, only IL-6 and IL-12 secretion became induced upon CpG ODN stimulation. We conclude that monocytes, but not B or T cells, represent the prime source of cytokines. Monocytes up-regulated expression of antigen-presenting, major histocompatibility complex class I and class II molecules in response to CpG DNA. In addition, both monocytes and B cells up-regulate costimulatory CD86 and CD40 molecules. The activation by CpG ODN depended on sequence motifs containing the core dinucleotide CG since destruction of the motif strongly reduced immunostimulatory potential. [ABSTRACT FROM AUTHOR]