Dov L. Boros

Soluble egg antigen-stimulated T helper lymphocyte apoptosis and evidence for cell death mediated by FasL(+) T and B cells during murine Schistosoma mansoni infection.

ABSTRACT Granuloma formation around schistosomal eggs is induced by soluble egg antigens (SEA) and mediated by the activity of CD4+ Th lymphocytes and their cytokines. Regulation of the inflammatory Th cell response during infection is still insufficiently understood. The hypothesis of this study was that activation-induced cell death (AICD) of CD4+ T cells is involved in the immune inflammatory response. This study investigated the dynamics of splenic and granuloma CD4+ Th cell apoptosis and Fas ligand (FasL) expression during the acute and chronic stages of murine schistosomal infection. Enhanced apoptosis of freshly isolated CD4+ Th lymphocytes commenced after egg deposition and persisted during the peak and modulated phases of granuloma formation. After oviposition, CD4+, CD8+, and CD19+ splenocytes and granuloma cells expressed elevated levels of FasL but FasL expression declined during the downmodulated stage of infection. In culture, SEA induced splenic and granuloma CD4+ T-cell apoptosis and stimulated expression of FasL on splenic but not granuloma CD4+ T cells, CD8+ T cells, and CD19+ B cells. SEA-stimulated splenocytes and granuloma cells preferentially lysed a Fas-transfected target cell line. Depletion of B cells from SEA-stimulated splenic cultures decreased CD4+ T cell apoptosis. Coculture of purified splenic B cells with CD4+ T cells and adoptive transfer of purified B cells indicated that antigen-stimulated B cells can kill CD4+ Th cells. However, CD4+ T cells were the dominant mediators of apoptosis in the granuloma. This study indicates that AICD is involved in the apoptosis of CD4+ T cells during schistosomal infection.

Fas Ligand-Expressing B-1a Lymphocytes Mediate CD4+-T-Cell Apoptosis during Schistosomal Infection: Induction by Interleukin 4 (IL-4) and IL-10

ABSTRACT A previous study of the murine model of Schistosoma mansoni infection has implicated splenic CD19+ B lymphocytes as Fas ligand (FasL)-bearing mediators of CD4+ T-lymphocyte apoptosis. The present study shows that B-cell deficiency leads to decreased CD4+ T-cell apoptosis during infection and compares FasL expression and killer function of B-1a- and CD5− B-lymphocyte subsets. B-1a cells from uninfected mice displayed constitutive expression of FasL compared with that of CD5− B cells. FasL expression was enhanced following worm egg deposition and antigenic stimulation on both subsets of B cells. Purified B-1a cells from uninfected mice were potent effectors of CD4+ T-cell apoptosis, and the killing effect was enhanced during schistosome infection. FasL expression by splenic B cells required CD4+-T-cell help that was replaced by addition of culture supernatants from antigen-stimulated splenocytes of infected mice. The culture-supernatant-stimulated FasL expression was inhibited by anti-interleukin 10 (IL-10) and anti-IL-4 antibodies. Culture of purified B cells with recombinant IL-4 (rIL-4), rIL-10, and soluble egg antigens (SEA) led to increased expression of FasL on B-1a cells. These results suggest that FasL-expressing, splenic B-1a cells are important mediators of SEA-stimulated CD4+-T-cell apoptosis and that maximal FasL expression on B-1a cells is dependent on antigenic stimulation and the presence of IL-4 and IL-10.

Retroviral Foxp3 gene transfer ameliorates liver granuloma pathology in Schistosoma mansoni infected mice

Schistosomiasis mansoni, a tropical helminthic disease, is caused by disseminated worm eggs that induce CD4+ T‐cell mediated granulomatous inflammation and fibrosis. T suppressor cell activity has been proposed as one of the mechanisms active in the down‐modulation of the murine disease during the chronic stage (16–20 weeks of the infection). In recent years a new category of the CD4+ CD25+ T regulatory (Treg) lymphocyte has been identified that maintains immune tolerance to self, and also functions in the regulation of parasite‐induced immunopathology. The Foxp3 gene which encodes the transcription factor Scurfin was found to be expressed by and required for the generation of CD4+ CD25+ T reg. At 8 weeks of the infection Foxp3 gene expression of splenocytes was similar to that of naïve mice, but increased fourfold by 16 weeks. In contrast, granulomatous livers at 8 and 16 weeks showed 10‐ and 30‐fold increases, respectively, in gene expression compared with normal liver. The percentage of granuloma CD4+ CD25+ T cells rose from 12% at 8 weeks to 88% at 16 weeks of the infection. Foxp3 expression was 3·5‐fold higher in the CD4+ CD25+ versus the CD4+ CD25– T cells in the 8 week infection granulomas. As a novel observation neuropilin‐1 membrane expression, a recently identified marker for Treg, was correlated with Foxp3 expression in the granuloma CD4+ CD25+ but not the CD25– cells. Co‐incubation with polyclonal stimulation of CD4+ CD25+ splenic cells with CD4+ CD25– cells suppressed proliferation of the latter. Retroviral transfer of the Foxp3 gene at the onset of granuloma formation enhanced fourfold Foxp3 expression in the granuloma CD4+ CD25+ T cells and strongly suppressed full granuloma development. Gene transfer also significantly enhanced transforming growth factor‐β, interferon‐γ and interleukin‐4 but not interleukin‐10 expression. It is concluded, that CD4+ CD25+, Foxp3+ Treg cells also regulate schistosome egg‐induced immunopathology.

The Role of Cytokines in the Formation of the Schistosome Egg Granuloma

Schistosomiasis mansoni is a helminth-induced disease infecting over 120 million people in the tropics. Morbidity and mortality are caused by parasite eggs that evoke in the liver and intestines of infected persons, T cell-mediated granulomatous inflammation and irreversible fibrosis. In the murine model granulomatous inflammation is induced by CD4+ T helper lymphocytes. This short review summarizes recent observations that implicate a variety of lymphokines and cytokines as mediators of the granulomatous inflammatory response. Mediator production was examined in splenocyte as well as granuloma cell cultures of infected or egg granuloma-bearing mice. In the synchronous pulmonary granuloma model generated around i.v. injected eggs in naive mice IL-1 mRNA expression and IL-1 production were detectable within the first 4 days of granuloma growth. After 4-6 days TNF-alpha mRNA message appeared and cytokine production was observed. With the aging of the granuloma, production of both cytokines diminished. Thus, these cytokines are considered to be the primary recruiters of cellular aggregation in granuloma growth. The role of TNF-alpha in granuloma formation was also confirmed in infected mice. Whereas treatment of animals with anti-TNF-alpha antiserum diminished hepatic granuloma size, repeated injection of murine rTNF-alpha into chronically-infected mice enhanced the downmodulated granuloma response. With the administration of specific anti-lymphokine mAbs and recombinant murine lymphokines, as well as serial assays of lymphokine production by splenic, granuloma lymphocytes of infected mice, the role of INF-gamma, IL-2 and IL-4 was delineated. Interferon-gamma was found to be produced very early at the inception of the liver granulomatous response. By the time granulomas reached maximal size (8 wks post infection) production declined. Concurrently IL-2, IL-4 production peaked with maximal granuloma growth and declined with the onset of the immune modulation of the inflammation. Whereas these latter lymphokines appear to play a proinflammatory role, IFN-gamma when administered in large doses diminished granulomatous inflammation, plays a regulatory role in the maintenance of the granulomatous response. The T helper cell population of the granulomas may also influence the lymphokine profile of the developing granuloma. So far precursor type TH0, and TH2 subset of helper cells have been cloned from liver granulomas. The former secreted both IL-2, IL-4 and IFN-gamma lymphokines and adoptively transferred the granulomatous response.(ABSTRACT TRUNCATED AT 400 WORDS)

Soluble Egg Antigens from Schistosoma mansoni Induce Angiogenesis-Related Processes by Up-Regulating Vascular Endothelial Growth Factor in Human Endothelial Cells

Schistosomiasis mansoni is characterized by hepatic granuloma formation. Endothelial cell activation within these granulomas may contribute to their development and to increased vascularization in the granuloma periphery. The earliest event in granuloma formation is the lodging of schistosome eggs within presinusoidal capillaries. The eggs secrete factors that may activate endothelial cells. This study investigated the effects of Schistosoma mansoni soluble egg antigen (SEA) on angiogenic processes: proliferation, tube formation, and apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs require serum and growth factors to proliferate in vitro. Proliferation occurred when SEA or live eggs were substituted for growth factors, but not for serum. SEA increased HUVEC tube formation and decreased HUVEC apoptosis after serum and growth factor deprivation. Messenger RNA for vascular endothelial growth factor (VEGF) increased 2-fold in SEA-treated HUVECs. These findings suggest that products secreted by schistosome eggs may promote angiogenesis within hepatic granulomas by up-regulating endothelial cell VEGF.

Expression of matrix metalloproteinases and their inhibitors during the resorption of schistosome egg-induced fibrosis in praziquantel-treated mice

Schistosomiasis mansoni is a tropical helminthic disease characterized by parasite egg‐induced granulomatous inflammation and cumulative fibrosis. Because fibrosis is influenced by the imbalance between degradative matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), we analysed the resorption of fibrous tissue and MMP/TIMP expression in the livers of S. mansoni‐infected and praziquantel‐cured mice. Worm elimination significantly enhanced survival rate, ameliorated the granulomatous pathology and reduced collagen I, III and IV gene expression at 6 and 12 months post‐treatment. Compared to 6 months infected, untreated controls, liver fibrous tissue was resorbed by 71·4% at 12 months after treatment. At 3 months post‐treatment, expression of the MMP‐2, ‐3, ‐8, ‐10, ‐13, ‐14 and ‐16 genes decreased compared with untreated controls. By 6 months, a highly significant increase in MMP‐10 gene expression was manifest. At 12 months, messages for all MMP genes decreased in relation to untreated controls. TIMP‐1, ‐2 and ‐3 gene expression drastically decreased between 3 and 6 months. At 1 year, only TIMP‐1 expression was significantly diminished. Overall, profibrogenic tumour necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β and inducible nitric oxide synthase (iNOS) gene expression decreased. Antigen‐stimulated splenocytes secreted significantly higher levels of interleukin (IL)‐4, IL‐5, IL‐10 and IL‐13 cytokines between 3 and 12 months after treatment. Production of interferon (IFN)‐γ was higher than in untreated controls 3 and 6 months after treatment. In conclusion, praziquantel‐treated mice showed a slow resorption of liver fibrous tissue. Resorption is attributed to the precipitous drop in TIMP‐1 gene expression level, which shifted the balance in favour of MMP message expression and presumed enhanced collagenase activity.

Immunoregulation of Granuloma Formation in Murine Schistosomiasis Mansoni

Schistosomiasis mansoni is a chronic, T lymphocyte-mediated granulomatous disease that affects mainly the liver and intestines of the infected host. The chronic inflammatory process and subsequent fibrous repair are the major factors in the pathology of the disease. In the murine model of schistosomiasis at the onset of the chronic stage of the infection, the granulomatous response undergoes spontaneous modulation with concomitant alleviation of the pathologic disturbance. Analysis of the process of modulation revealed that it results from the interaction between inflammatory and regulatory subpopulations of T lymphocytes. At the acute phase of the infection, the granulomatous response is initiated and maintained by inflammatory TDH cells that release lymphokines which mobilize and recruit the macrophages, eosinophils, etc. for the generation of the lesion. Already at this stage, while the inflammatory influence prevails, a low number of suppressor T lymphocytes are present. With the progress of the infection, the overheated inflammatory response is curbed by regulatory processes. At least two, but perhaps more, T suppressor lymphocytes are involved in the maintenance of the modulation of the granulomatous response. Modulation is an active process that needs constant maintenance, probably by recruitment of fresh suppressor cells. Removal of the suppressor population causes an immediate elevation of the granulomatous response. During modulation, T suppressor lymphocytes either abrogate or greatly diminish inflammatory lymphokine production. This in turn may be the cause for decreased cell recruitment and diminution in newly formed granuloma size. Apparently a total abrogation of the granulomatous response is not desirable because released egg antigens can be harmful to liver parenchyma cells. This has been demonstrated both in thymus-deprived and in nude, infected mice. Thus, a smaller inflammatory response has the double advantage of not only being less destructive, but also shielding the underlying tissue from damage by parasite products. The various subpopulations of T lymphocytes communicate with one another by means of soluble suppressor factors that arise from the suppressor T cells. The factors may have different functions. One factor may regulate lymphokine production whereas another may recruit fresh suppressor cells from a pool of precursors. The factors act in an antigen-specific manner. Tentatively, one may assume that these factors are composed of two units: one is the I subregion membrane marker and the other is the specific recognition receptor. The nature of this receptor is still unclear, but it may be an anti-idiotypic determinant.(ABSTRACT TRUNCATED AT 400 WORDS)

Dynamics of collagen, MMP and TIMP gene expression during the granulomatous, fibrotic process induced by Schistosoma mansoni eggs.

Abstract In schistosomiasis mansoni, granulomatous inflammation and fibrotic resolution are the major pathogenetic factors. The outcome of fibrosis is influenced by the deposition of collagen and degradation mediated by matrix metalloproteinases (MMP). There is a dearth of data on the expression of MMP and the tissue inhibitors of metalloproteinase (TIMP) during the fibrosis associated with schistosomiasis. In this study, the dynamics of collagen, MMP and TIMP gene expression were analysed during murine Schistosoma mansoni infection. Expression within the granulomatous liver tissue of the genes coding for collagen of types I, III and IV was up-regulated at the onset of granuloma development, and the dominant type-I expression peaked at the chronic, fibrotic stage. The amount of deposited hepatic collagen increased with the chronicity of the infection, indicating cumulative fibrosis. Collagenase, gelatinase, stromelysin, matrilysin-specific gene activities were similarly up-regulated, but only MMP-8 (collagenase-2) expression peaked at the height of fibrosis. TIMP-1 gene expression gradually increased during the course of the disease and, along with TIMP-2, peaked at the chronic, fibrotic stage. Granuloma myofibroblasts expressed both MMP and TIMP-1 genes. In ELISA of the splenic cytokines, high levels of fibrogenic interleukin-13 and moderate production of transforming growth factor- b were found to be concurrent with fibrosis. These data indicate that an imbalance in MMP:TIMP expression and fibrogenic cytokine production are associated with cumulative fibrosis.

Molecular mimicry and horror autotoxicus : do chlamydial infections elicit autoimmunity?

All species of the order Chlamydiales are obligate intracellular eubacterial pathogens of their various hosts. Two chlamydial species, Chlamydia trachomatis and Chlamydia pneumoniae, are primarily human pathogens, and each is known to cause important diseases. Some strains of C. trachomatis are sexually transmitted and frequently cause severe reproductive problems, primarily in women. Other strains of the organism serve as the aetiological agents for blinding trachoma, still the leading cause of preventable blindness in underdeveloped nations. C. pneumoniae is a respiratory pathogen known to cause community-acquired pneumonia. Importantly, both organisms engender an immunopathogenic response in the human host, and both have been associated with widely diverse, relatively common and currently idiopathic chronic diseases, most of which include an important autoimmune component. In this article, we explore the available experimental data regarding the possible elicitation of autoimmunity in various contexts by chlamydial infection, and we suggest several avenues for research to explore this potentially important issue further.

The role of egg antigens, cytokines in granuloma formation in murine schistosomiasis mansoni

The induction of granuloma formation by soluble egg antigens (SEA) of Schistosoma mansoni is accompanied by T cell-mediated lymphokine production that regulates the intensity of the response. In the present study we have examined the ability of SDS-PAGE fractionated SEA proteins to elicit granulomas and lymphokine production in infected and egg-immunized mice. At the acute stage of infection SEA fractions (< 21, 25-30, 32-38, 60-66, 70-90, 93-125, and > 200 kD) that elicited pulmonary granulomas also elicited IL-2, IL-4 lymphokine production. At the chronic stage a diminished number of fractions (60-66, 70-90, 93-125, and > 200 kD) were able to elicit granulomas with an overall decrease in IL-2, IL-4 production. Granulomas were elicited by larval-egg crossreactive and egg-specific fractions at both the acute and chronic stage of the infection. Examination of lymphokine production from egg-immunized mice demonstrated that as early as 4 days IL-2 was produced by spleen cells stimulated with < 21, 32-38, 40-46, 93-125, and > 200 kD fractions. By 16 days, IL-2 production was evoked by 8 of 9 fractions. IL-4 production at 4 days in response to all fractions was minimal while at 16 days IL-4 was elicited with the < 21, 25-30, 50-56, 93-125, and > 200 kD fractions. The present study reveals differences in the range of SEA fractions able to elicit granulomas and IL-2, IL-4 production between acute and chronic stages of infection.(ABSTRACT TRUNCATED AT 250 WORDS)