Horst Mossmann

Alternative Macrophage Activation Is Essential for Survival during Schistosomiasis and Downmodulates T Helper 1 Responses and Immunopathology

Macrophage/neutrophil-specific IL-4 receptor alpha-deficient mice (LysM(Cre)IL-4Ralpha(-/flox)) were generated to understand the role of IL-4/IL-13 responsive myeloid cells during Type 2 immune responses. LysM(Cre)IL-4Ralpha(-/flox) mice developed protective immunity against Nippostrongylus brasiliensis accompanied by T(H)2 development and goblet cell hyperplasia. In contrast, LysM(Cre)IL-4Ralpha(-/flox) mice were extremely susceptible to Schistosoma mansoni infection with 100% mortality during acute infection. Mortality was not dependent on neutrophils and occurred in the presence of T(H)2/Type 2 responses, granuloma formation, and egg-induced fibrosis. Death was associated with increased T(H)1 cytokines, hepatic and intestinal histopathology, increased NOS-2 activity, impaired egg expulsion, and sepsis. IL-10 was not able to compensate for the absence of IL-4/IL-13-activated alternative macrophages. Together, this shows that alternative macrophages are essential during schistosomiasis for protection against organ injury through downregulation of egg-induced inflammation.

Functional testosterone receptors in plasma membranes of T cells

T cells are considered to be unresponsive to testosterone due to the absence of androgen receptors (AR). Here, we demonstrate the testosterone responsiveness of murine splenic T cells in vitro as well as the presence of unconventional cell surface receptors for testosterone and classical intracellular AR. Binding sites for testosterone on the surface of both CD4+ and CD8+ subsets of T cells are directly revealed with the impeded ligand testosterone‐BSA‐FITC by confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. Binding of the plasma membrane impermeable testosterone‐BSA conjugate induces a rapid rise (<5s) in[Ca2+]i of Fura‐2‐loaded T cells. This rise reflects influx of extracellular Ca2+ through non‐voltage‐gated and Ni2+‐blockable Ca2+ channels of the plasma membrane. The testosterone‐BSA‐induced Ca2+ import is not affected by cyproterone, a blocker of the AR. In addition, AR are not detectable on the surface of intact T cells when using anti‐AR antibodies directed against the amino and carboxy terminus of the AR, although T cells contain AR, as revealed by reverse transcription‐polymerase chain reactions and Western blotting. AR can be visualized with the anti‐AR antibodies in the cytoplasm of permeabilized T cells by using CLSM, though AR are not detectable in cytosol fractions when using the charcoal binding assay with 3H‐R1881 as ligand. Cytoplasmic AR do not translocate to the nucleus of T cells in the presence of testosterone, in contrast to cytoplasmic AR in human cancer LNCaP cells. These findings suggest that the classical AR present in splenic T cells are not active in the genomic pathway. By contrast, the cell surface receptors for testosterone are in a functionally active state, enabling T cells a nongenomic response to testosterone.—Benten, W. P. M., Lieberherr, M., Giese, G., Wrehlke, C., Stamm, O., Sekeris, C. E., Mossmann, H., Wunderlich, F. Functional testosterone receptors in plasma membranes of T cells. FASEB J. 13, 123–133 (1999)

Containment of aerogenic Mycobacterium tuberculosis infection in mice does not require MyD88 adaptor function for TLR2, -4 and -9.

The role of Toll‐like receptors (TLR) and MyD88 for immune responses to Mycobacterium tuberculosis (Mtb) infection remains controversial. To address the impact of TLR‐mediated pathogen recognition and MyD88‐dependent signaling events on anti‐mycobacterial host responses, we analyzed the outcome of Mtb infection in TLR2/4/9 triple‐ and MyD88‐deficient mice. After aerosol infection, both TLR2/4/9‐deficient and wild‐type mice expressed pro‐inflammatory cytokines promoting antigen‐specific T cells and the production of IFN‐γ to similar extents. Moreover, TLR2/4/9‐deficient mice expressed IFN‐γ‐dependent inducible nitric oxide synthase and LRG‐47 in infected lungs. MyD88‐deficient mice expressed pro‐inflammatory cytokines and were shown to expand IFN‐γ‐producing antigen‐specific T cells, albeit in a delayed fashion. Only mice that were deficient for MyD88 rapidly succumbed to unrestrained mycobacterial growth, whereas TLR2/4/9‐deficient mice controlled Mtb replication. IFN‐γ‐dependent restriction of mycobacterial growth was severely impaired only in Mtb‐infected MyD88, but not in TLR2/4/9‐deficient bone marrow‐derived macrophages. Our results demonstrate that after Mtb infection neither TLR2, ‐4, and ‐9, nor MyD88 are required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, TLR4 and TLR9, is critical for triggering macrophage effector mechanisms central to anti‐mycobacterial defense.

Testosterone signaling in T cells and macrophages

This review summarizes data about non-genomic actions of testosterone on murine malaria, T cells and macrophages produced by our group during the last 15 years. In C57BL/10 mice, testosterone induces a lethal outcome of blood stage infections with Plasmodium chabaudi which normally takes a self-healing course controlled by genes of the H-2 complex and the non-H-2 background. This suppressive effect of testosterone is mediated neither via the classic intracellular androgen receptor (AR) response nor, after conversion of testosterone to estradiol, via the estrogen receptor. Testosterone acts non-genomically, i.e. through surface receptors, on murine T cells and macrophages, which becomes evident as a rapid rise in the intracellular free Ca(2+) concentration ([Ca(2+)](i)). In T cells, this rise reflects predominantly influx of extracellular Ca(2+), while it is predominantly due to release of Ca(2+) from intracellular Ca(2+)-stores in macrophages. The testosterone-induced rise in [Ca(2+)](i) of both macrophages and T cells is not inhibited by the AR-blocker cyproterone, and it is also inducible by the plasma membrane impermeable ligand testosterone-BSA. The surface receptors initiate a transcription-independent signaling pathway of testosterone. Currently, we are trying to isolate testosterone surface receptors and to investigate a possible cross-talk of non-genomic testosterone signaling with other genotropic signaling pathways.

IL-12-Independent IFN-γ Production by T Cells in Experimental Chagas’ Disease Is Mediated by IL-18

IL-12p35-deficient (IL-12p35−/−) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas’ disease. Delayed immune responses were observed in mutant mice, although comparable IFN-γ and TNF-α blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4+ T cells as the cellular source for IL-12-independent IFN-γ production. IL-18 was induced in IL-12p35−/− mice and was responsible for IFN-γ production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-γ production in experimental Chagas’ disease that is T cell and IL-18 dependent.

Testosterone and other gonadal factor(s) restrict the efficacy of genes controlling resistance to Plasmodium chabaudi malaria

Summary The effect of circulating concentrations of testosterone (Te) on resistance to Plasmodium chabaudi malaria was investigated in the H‐2 congenic mouse strains C57BL/1, B10.A. 10.A(3R). B10.A(4R), and B10.D2. Te‐levels were determined by radioimmunoassay and resistance was expressed in terms of percent self‐healers after challenge with 106P. chabaudi‐infected erythrocytes. Our data indicate: (i) Females and castrated males reveal very similar interstrain variations of resistance. These do not correlate with the interstrain variations of the Te‐levels. This is consistent with the view that resistance to P. chaubaudi is controlled by genes of the H‐2 complex and genes of the non‐H‐2 B10‐background, (ii) The polygenic control of resistance is inefficacious at high Te‐levels. This is evident as high susceptibilities of males, Te‐treated females and Te‐treated castrated males. Moreover, high Te‐levels correlate with susceptibilities to P. chabaudi within mice of the same sex of a given strain, (iii) BlO‐males chemically castrated using buserdin display the same low Te‐leveel as those surgically castrated. The latter become resistant, while the former remain as highly susceptible to P. chabaudi is untreated B10‐males. Obviously, other gonadal factor(s), besides Te, impose restrictions on genes controlling resistance to P. chabaudi malaria.

Concerted action of perforin and granzymes is critical for the elimination of Trypanosoma cruzi from mouse tissues, but prevention of early host death is in addition dependent on the FasL/Fas pathway

CTL and NK cells are critical for resistance to acute Trypanosoma cruzi infection, but are also implicated in the pathology induced by this intracellular protozoan parasite. Here we explore to what extent the two main cytolytic pathways of CTL and NK cells, i.e. the granule exocytosis and the Fas ligand (FasL)/Fas pathways, are responsible for the elimination of parasites from mouse tissues and control of organ pathology. For this purpose we have employed mouse strains with targeted gene defects in one or more components — including perforin, granzyme A and granzyme B, and Fas — of either of the two cytolytic pathways, and we used the highly pathogenic T. cruzi strain Tulahuen. We show that parasites are effectively cleared from infected tissues independently of the FasL/Fas pathway by the concerted action of perforin and the two granzymes. However, prevention of pathology and early host death is critically dependent in addition on an operational FasL/Fas interaction. Thus, in contrast to C57BL/6 (B6) wild‐type mice, mouse strains with deficiencies in either the FasL/Fas or the perforin/granzyme pathway similarly suffer from early death, independently of their differential capacity to control parasite growth; this finding indicates that the two cytolytic pathways control distinct but vital processes during infection with T. cruzi.

Testosterone Suppresses Protective Responses of the Liver to Blood-Stage Malaria

ABSTRACT Testosterone induces a lethal outcome in otherwise self-healing blood-stage malaria caused by Plasmodium chabaudi. Here, we examine possible testosterone effects on the antimalaria effectors spleen and liver in female C57BL/6 mice. Self-healing malaria activates gating mechanisms in the spleen and liver that lead to a dramatic reduction in trapping activity, as measured by quantifying the uptake of 3-μm-diameter fluorescent polystyrol particles. However, testosterone delays malaria-induced closing of the liver, but not the spleen. Coincidently, testosterone causes an ∼3- to 28-fold depression of the mRNA levels of nine malaria-responsive genes, out of 299 genes tested, only in the liver and not in the spleen, as shown by cDNA arrays and Northern blotting. Among these are the genes encoding plasminogen activator inhibitor (PAI1) and hydroxysteroid sulfotransferase (STA2). STA2, which detoxifies bile acids, is suppressed 10-fold by malaria and an additional 28-fold by testosterone, suggesting a severe perturbation of bile acid metabolism. PAI1 is protective against malaria, since disruption of the PAI1 gene results in partial loss of the ability to control the course of P. chabaudi infections. Collectively, our data indicate that the liver rather than the spleen is a major target organ for testosterone-mediated suppression of resistance against blood-stage malaria.

Massive Destruction of Malaria-Parasitized Red Blood Cells despite Spleen Closure

ABSTRACT It is currently accepted that malaria-parasitized red blood cells (pRBC) are eliminated, like senescent erythrocytes, phagocytically by macrophages in the red pulp of the spleen. Here, however, we show that self-healing Plasmodium chabaudi malaria activates spleen closure in C57BL/6 mice. Confocal laser scanning microscopy revealed that spleen closing was manifested by elimination of entry into the red pulp of 3-μm polystyrol particles, pRBC, and nonparasitized red blood cells but not of bovine serum albumin. This spleen closure did not reflect a reduction in the number of phagocytic cells, as shown by flow cytometry, whereas marginal zone macrophages (MZM) were lost and red pulp macrophages entered the white pulp. Splenic trapping of pBRC was strongly reduced in the absence of MZM and marginal metallophilic macrophages (MMM), as it is in noninfected mice with a disrupted lymphotoxin β receptor (LTβR−/−), and it was still significantly reduced when the number of MZM and MMM was diminished, as in tumor necrosis factor alpha-deficient (TNF-α−/−) mice. Moreover, mice deficient in TNF-α, tumor necrosis factor receptor I (TNFRI−/−), and LTβR exhibited progressive impairment in malaria-induced spleen closing. Treatment of C57BL/6 mice with TNF-α induced loss of MZM and spleen closing by about 20%. Our data indicate that TNF/TNFRI signaling is involved in regulating malaria-induced spleen closure, which is maximal during crisis, when parasitemia declines more than 100-fold. Consequently, the vast majority of pRBC cannot be destroyed by the spleen during crisis, suggesting that the known sophisticated sequestration system of Plasmodium parasites did not evolve to avoid splenic clearance.

Th2 cells shape the differentiation of developing T cell responses during interactions with dendritic cells in vivo

During priming, naive CD4+ Th cells differentiate into cells that produce either IFN‐γ or IL‐4. Even though the cascade of pathways that induces IL‐4‐producing Th2 cells has been determined in vitro, the signals promoting Th2 differentiation under physiological conditions remain enigmatic, especially the natural role of the single most important Th2‐inducing signal,IL‐4. Using Th2 and naive Th cells, each expressing a distinct transgenic TCR, here we show that Th2 cells migrate with the same dynamics as naive Th cells in draining lymph nodes and bind to the same DC, when driven by antigen in complete Freunds adjuvant (CFA). Th2‐cell‐derived IL‐4 deviates CFA‐induced Th1 development toward a Th2 phenotype, if both cell populations co‐localize in the same T cell area, and are activated simultaneously. Thus, intranodal Th2 cells directly influence Th cell differentiation in vivo, but only under restricted conditions. These findings have implications for the design of cytokine‐based therapies and explain the spreading of Th2 responses to multiple aeroallergens in allergic asthma, where naive Th and Th2 cells co‐localize in lung‐draining lymph nodes.