Martin Röllinghoff

The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4.

Macrophages release IFN-γ on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-γ by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-γ production in NK cells, was not required for IFN-γ production by these macrophages. IL-12 alone already induced the expression of IFN-γ mRNA, but nuclear translocation of STAT4, the release of IFN-γ protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-κB, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-β) also suppressed macrophage IFN-γ production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-γ by macrophages and point to a diversity in the signals required for IFN-γ production by various cell types.

Rapidly Fatal Leishmaniasis in Resistant C57BL/6 Mice Lacking TNF

The resolution of infections with the protozoan parasite Leishmania major in mice requires a Th1 response that is closely associated with the expression of IL-12, IFN-γ, and inducible NO synthase. Previous Ab neutralization studies or the use of mice deficient for both TNF receptors suggested that TNF plays only a limited role in the control of parasite replication in vivo. In this study we demonstrate that resistant C57BL/6 (B6.WT) mice locally infected with L. major rapidly succumb to progressive visceral leishmaniasis after deletion of the TNF gene by homologous recombination. A reduction of the parasite inoculum to 3000 promastigotes did not prevent the fatal outcome of the disease. An influence of the altered morphology of secondary lymphoid organs in C57BL/6-TNF−/− (B6.TNF−/−) mice on the course of disease could be excluded by the generation of reciprocal bone marrow chimeras. Although infected B6.TNF−/− mice mounted an L. major-specific IFN-γ response and expressed IL-12, the onset of the immune reaction was delayed. After in vitro stimulation, B6.TNF−/− inflammatory macrophages released 10-fold less NO in response to IFN-γ than B6.WT cells. However, in the presence of a costimulus, e.g., L. major infection or LPS, the production of NO by B6.WT and B6.TNF−/− macrophages was comparable. In vivo, inducible NO synthase protein was readily detectable in skin lesions and draining lymph nodes of B6.TNF−/− mice, but its expression was more disperse and less focal in the absence of TNF. These are the first data to demonstrate that TNF is essential for the in vivo control of L. major.

High Diversity of ankA Sequences of Anaplasma phagocytophilum among Ixodes ricinus Ticks in Germany

ABSTRACT In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were ≥99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.

Lymphocytes play the music but the macrophage calls the tune

Researchers interested in immunological aspects of bacterial, fungal, protozoan and helminthic infection are too often kept apart by artificial subject boundaries. These barriers were temporarily breached by a recent workshop* in which the complex interplay between microbes and their mammalian hosts were examined from a global viewpoint. The role of T-cell subsets and their products came under close scrutiny but the most forceful image was that of the macrophage. As host for infective agents, as modulator of specific immune activity and as ultimate mediator of the host response, the macrophage plays a virtuosos role in the host-parasite drama.

Expression of inducible nitric oxide synthase in skin lesions of patients with american cutaneous leishmaniasis

ABSTRACT Cytokine-inducible (or type 2) nitric oxide synthase (iNOS) is indispensable for the resolution of Leishmania major or Leishmania donovani infections in mice. In contrast, little is known about the expression and function of iNOS in human leishmaniasis. Here, we show by immunohistological analysis of skin biopsies from Mexican patients with local (LCL) or diffuse (DCL) cutaneous leishmaniasis that the expression of iNOS was most prominent in LCL lesions with small numbers of parasites whereas lesions with a high parasite burden (LCL or DCL) contained considerably fewer iNOS-positive cells. This is the first study to suggest an antileishmanial function of iNOS in human Leishmania infections in vivo.

Interleukin-10 Inhibits Antimicrobial Activity Against Leishmania major in Murine Macrophages

The stimulation of macrophages is of importance to the defense against intracellularly replicating microorganisms such as Leishmania. In this study the direct effect of recombinant interleukin‐10 (IL‐10) on the leishmanicidal effector functions of murine peritoneal or bone marrow derived macrophuges was investigated. IL‐10 almost completely inhibited the killing of intracellular leishmania at concentrations above 10ng/ml. This inhibitory effect was independent of the stimulus used as the activation of macrophages by IFN‐γ and IL‐7, recently shown to possess macrophage activating properties, were suppressed by IL‐10. Kinetic experiments revealed that IL‐10 must be present during the process of macrophage activation and that the leishmanicidal effector function of fully activated macrophages was not influenced. Furthermore, in the absence of exogenously added IL‐10, the addition of neutralizing antibodies against IL‐10 or IL‐10‐specific antisense phosphorothioate DNA‐oligonucleotide led to an enhanced killing of parasites after stimulation with either IFN‐7 or IL‐7. In accordance with this, IL‐10 mRNA was readily detectable in murine macrophages by PCR with reverse transcribed mRNA. These results indicate that IL‐10, which is endogenously produced by macrophages, acts as an autocrine deactivating factor supporting the survival of the parasite.

High Levels of Susceptibility and T Helper 2 Response in MyD88-Deficient Mice Infected with Leishmania major Are Interleukin-4 Dependent

ABSTRACT Myeloid differentiation protein 88 (MyD88) is a general adaptor for the signaling cascade through receptors of the Toll/IL-1R family. When infected with Leishmania major parasites, MyD88-deficient mice displayed a dramatically enhanced parasite burden in their tissues similar to that found in susceptible BALB/c mice. In contrast, MyD88 knockout mice did not develop ulcerating lesions despite a lack of interleukin-12 (IL-12) production and a predominant T helper 2 cell response. Blockade of IL-4 produced early (day 1) after infection restored a protective T helper 1 response in MyD88 knockout mice.

Protection against Progressive Leishmaniasis by IFN-β

Type I IFNs (IFN-αβ) exert potent antiviral and immunoregulatory activities during viral infections, but their role in bacterial or protozoan infections is poorly understood. In this study, we demonstrate that the application of low, but not of high doses of IFN-β protects 60 or 100% of BALB/c mice from progressive cutaneous and fatal visceral disease after infection with a high (106) or low (104) number of Leishmania major parasites, respectively. IFN-β treatment of BALB/c mice restored the NK cell cytotoxic activity, increased the lymphocyte proliferation, and augmented the production of IFN-γ and IL-12 in the draining lymph node. Low, but not high doses of IFN-β caused enhanced tyrosine phosphorylation of STAT1 and STAT4, suppressed the levels of suppressor of cytokine signaling-1, and up-regulated the expression of inducible NO synthase in vivo. The IFN-β-induced increase of IFN-γ production was dependent on STAT4. Protection by IFN-β strictly required the presence of inducible NO synthase. In the absence of STAT4 or IL-12, IFN-β led to an amelioration of the cutaneous and visceral disease, but was unable to prevent its progression. These results identify IFN-β as a novel cytokine with a strong, dose-dependent protective effect against progressive cutaneous leishmaniasis that results from IL-12- and STAT4-dependent as well as -independent events.

Impact of Thymus on the Generation of Immunocompetence and Diversity of Antigen-Specific MHC-Restricted Cytotoxic T-Lymphocyte Precursors

The thymus represents the primary iymphoid organ producing (thymus derived) lymphocytes. Independent of antigenic stimulation, immunocompetent Tlymphocytes develop within this organ from prethymic precursor cells, which in turn are derived from hemopoetic stem cells. Thus, one ofthe prime roles ofthe thymus is to provide the microenvironment necessary for maturation and differentiation of prethymic T-precursor cells into mature T-cells, which are subsequently exported as post-thymic T-lymphocytes. In addition, production of one (or several) thytnic mediator(s), probably active at distinct sites, appears to be essential for maturation of immunocompetent T-cells (for review see Miller 1979). Not only the thymus, but also gene products of the MHC exert a profound influence on T-cell reactivity. It has been shown that the frequency of alloreactive T-lymphocytes is at least 100 times as high as the frequency of cells reacting to foreign antigens (Simonson 1967, Fischer-Lindahl & Wilson 1977, MacDonald et al. 1980). To account for this high frequency, Jerne (1971) originally proposed that the repertoire of T-cells depends on a set of germ-line vgenes which code for structures essentially complementary to the MHC-alleles of that species. In the thymus, the T-cell precursors that are specific for selfMHC gene products are first selected by self-MHC-antigens encountered on thymic epithehal cells and allowed to differentiate and to proliferate. In a second

Inverse Correlation of Maturity and Antibacterial Activity in Human Dendritic Cells

Dendritic cells (DCs) are a key part of host defense against microbial pathogens, being part of the innate immune system, but also instructing the adaptive T cell response. This study was designed to evaluate whether human DCs directly contribute to innate immunity by killing intracellular bacteria, using tuberculosis as a model. DCs were detected in bronchoalveolar lavage samples indicating that DCs are available for immediate interaction with Mycobacterium tuberculosis (M. Tb) after inhalation of the pathogen. The phenotype of DC in bronchoalveolar lavage closely resembles monocyte-derived immature DC (iDC) according to the expression of CD1a, CD83, and CCR7. The antimicrobial activity of iDC against intracellular M. Tb inversely correlated with TNF-α-release and was enhanced by treatment with anti-TNF-α Abs. Differentiation of iDC into mature DC by addition of TNF-α or activation via Toll-like receptors further reduced killing of M. Tb. The antibacterial activity against intracellular M. Tb of all DCs was significantly lower than alveolar macrophages. Therefore, the maintenance of a pool of DCs at the site of disease activity in tuberculosis, and the maturation of these DC by TNF-α provides a mechanism by which M. Tb escapes the innate immune system.