Mark P. Hedger

Activin A is a critical component of the inflammatory response, and its binding protein, follistatin, reduces mortality in endotoxemia

Activin A is a member of the transforming growth factor-β superfamily, which we have identified as having a role in inflammatory responses. We show that circulating levels of activin increase rapidly after LPS-induced challenge through activation of Toll-like receptor 4 and the key adaptor protein, MyD88. Treatment with the activin-binding protein, follistatin, alters the profiles of TNF, IL-1β, and IL-6 after LPS stimulation, indicating that activin modulates the release of several key proinflammatory cytokines. Further, mice administered one 10-μg dose of follistatin to block activin effects have increased survival after a lethal dose of LPS, and the circulating levels of activin correlate with survival outcome. These findings demonstrate activin As crucial role in the inflammatory response and show that blocking its actions by the use of follistatin has significant therapeutic potential to reduce the severity of inflammatory diseases.

Cytokines and the immune-testicular axis

Cytokines are regulatory proteins involved in haematopoiesis, immune cell development, inflammation and immune responses. Several cytokines have direct effects on testicular cell functions, and a number of these are produced within the testis even in the absence of inflammation or immune activation events. There is compelling evidence that cytokines, in fact, play an important regulatory role in the development and normal function of the testis. Pro-inflammatory cytokines including interleukin-1 and interleukin-6 have direct effects on spermatogenic cell differentiation and testicular steroidogenesis. Stem cell factor and leukaemia inhibitory factor, cytokines normally involved in haematopoiesis, also play a role in spermatogenesis. Anti-inflammatory cytokines of the transforming growth factor-beta family are implicated in testicular development. Consequently, local or systemic up-regulation of cytokine expression during injury, illness or infection may contribute to the disruption of testicular function and fertility that frequently accompanies these conditions. The aim of this review is to provide a very brief summary of the extensive literature dealing with cytokines in testicular biology, and to follow this with some speculation concerning the significance of these molecules in interactions between the immune system and the testis.

Macrophages and the immune responsiveness of the testis.

Immune responses within the testis are regulated in a manner that provides protection for the developing male germ cells, while permitting qualitatively normal inflammatory responses and protection against infection. The large population of resident-type macrophages in the testis is strongly implicated in mediating this specialised immunological environment. Several studies in the rat have shown that testicular macrophages retain their cytotoxic and phagocytic capacity, but have greatly diminished pro-inflammatory function and even exhibit immunosuppressive activity. While the local mechanisms that control the phenotype of the testicular macrophage population are unknown, evidence points to the influence of the testicular somatic cells, the Sertoli and Leydig cells. A smaller but significant population of macrophages that lack expression of resident macrophage markers, is also found in the rat testis. The functional role of these macrophages remains to be defined, but they most likely represent circulating monocytes or newly-arrived testicular macrophages, and, therefore, may contribute to sustaining inflammatory responses within the testis. Further investigation of the immune-related functions of these different macrophage subsets, and the testicular somatic cells, during immunological and inflammatory events should provide a better understanding of how the testicular immune environment is maintained and regulated.

Bacterial lipopolysaccharide-induced inflammation compromises testicular function at multiple levels in vivo.

While it is well known that serious illness and inflammation reduce male fertility, the mechanisms involved are poorly understood. In adult male rats, a single injection of lipopolysaccharide at doses that induced either mild or severe inflammation, caused a biphasic decline in Leydig cell testosterone production and gonadotropin responsiveness. In the high dose group only, serum LH levels also were reduced; however, intratesticular testosterone concentrations remained at a level adequate to support qualitatively normal spermatogenesis in both treatment groups. Testicular interstitial fluid formation also declined in a dose-dependent fashion after lipopolysaccharide treatment. In the high dose group only, these hormonal and vascular changes were accompanied by an increase in endothelial permeability, microhemorrhage, and inflammatory cells in the testis, followed by vacuolization of round spermatid nuclei, disruption of Sertoli-germ cell contacts at stages I–IV of the cycle of the seminiferous epithelium,...

Activin and related proteins in inflammation: Not just interested bystanders

Activin A, a member of the transforming growth factor-beta superfamily, is released rapidly into the circulation during inflammation. This review examines the evidence that activin is a critical mediator of inflammation and immunity. Activin modulates several aspects of the inflammatory response, including release of pro-inflammatory cytokines, nitric oxide production and immune cell activity. Crucially, inhibiting activin with follistatin, a high affinity binding protein, alters the pattern of cytokines released and improves survival in a mouse model of endotoxic shock. Serum and tissue concentrations of activin are elevated in a wide range of pathological conditions. The utility of activin as a diagnostic marker of clinical inflammation and the use of follistatin to block activin actions therapeutically are also discussed.

Inhibin and activin regulate [3H]thymidine uptake by rat thymocytes and 3T3 cells in vitro.

[3H]Thymidine incorporation by adult rat thymocytes, in the presence of phytohaemagglutinin (PHA), was stimulated by bovine inhibin (ED50 0.7 nM), and inhibited by bovine activin (ID50 0.4 nM) and porcine transforming growth factor-beta (TGF-beta) (ID50 4 pM); inhibin opposed the actions of activin and TGF-beta. Bovine 35 kDa follicle stimulating hormone (FSH) suppressing protein (FSP) had no effect on either unstimulated or PHA-stimulated thymocytes. Inhibin also stimulated thymocytes in the presence of a submaximal dose of concanavalin A (ConA), and in the absence of either lectin. Thymocytes which had been maximally stimulated by ConA were inhibited by TGF-beta (ID50 0.02 nM), but not affected by inhibin and activin. Both activin and TGF-beta stimulated [3H]thymidine uptake by 3T3 fibroblasts, but inhibin and FSP had no effect, alone or on activin-stimulated 3T3 fibroblasts. The results indicate that inhibin and activin have opposing, cell type-specific effects on the proliferation of T-lymphocytes, while activin also stimulates fibroblast proliferation in vitro.

Evidence for activin A and follistatin involvement in the systemic inflammatory response

The inflammatory cascade is a multifactorial process regulated by interwoven cytokine and growth factor networks. This review summarizes the emerging evidence that implicate activin A and follistatin in inflammatory processes. Our recent studies have determined that activin A is released early in the cascade of circulatory cytokines during systemic inflammatory episodes, roughly coincident with tumour necrosis factor (TNF)-alpha and before interleukin (IL)-6 and follistatin. The source(s) of this activin A are not yet established, but prime candidates are monocytes/macrophages, other immune cell types or vascular endothelial cells. Clinical data are limited, but activin beta(A) subunit mRNA or activin A protein is elevated in inflammatory bowel diseases and inflammatory arthropathies, and circulating concentrations of follistatin are elevated in patients with sepsis. In more mechanistic approaches, in vitro studies show that activin A can have both pro- and anti-inflammatory actions on key inflammatory mediators such as TNFalpha, IL-1beta and IL-6. Furthermore, there is emerging understanding of how the intracellular signaling pathway for activin A, incorporating Smads, may interact with and be modulated by other key regulatory cytokines and growth factors.

Inducible Nitric Oxide Synthase in the Rat Testis: Evidence for Potential Roles in Both Normal Function and Inflammation-Mediated Infertility

Abstract In vitro data have indicated that nitric oxide (NO) inhibits Leydig cell testosterone production, suggesting that NO may play a role in the suppression of steroidogenesis and spermatogenic function during inflammation. Consequently, we investigated expression of the inflammation-inducible isoform of NO synthase (iNOS) in the inflamed adult rat testis and the ability of a broad-spectrum inhibitor of NO production, l-nitro-l-arginine methyl ester, to prevent Leydig cell dysfunction during inflammation. Unexpectedly, immunohistochemical and mRNA data established that iNOS is expressed constitutively in Leydig cells and in a stage-specific manner in Sertoli, peritubular, and spermatogenic cells in the normal testis. Expression was increased in a dose-dependent manner in all these cell types during lipopolysaccharide (LPS)-induced inflammation. In noninflamed testes, treatment with the NO synthase inhibitor reduced testicular interstitial fluid formation and testosterone production without any effect on serum LH levels. Administration of the inhibitor did not prevent the suppression of testicular interstitial fluid and testosterone production that occurs within 6 h after LPS treatment. Collectively, these data indicate a novel role for iNOS in autocrine or paracrine regulation of the testicular vasculature, Leydig cell steroidogenesis, and spermatogenesis in the normal testis. The data suggest that increased NO is not the major cause of acute Leydig cell dysfunction in the LPS-treated inflammation model, although a role for NO in this process cannot be excluded, particularly at other time points. Moreover, up-regulation of iNOS may contribute to the seminiferous epithelium damage caused by LPS-induced inflammation.

The regulation and functions of activin and follistatin in inflammation and immunity

The activins are members of the transforming growth factor β superfamily with broad and complex effects on cell growth and differentiation. Activin A has long been known to be a critical regulator of inflammation and immunity, and similar roles are now emerging for activin B, with which it shares 65% sequence homology. These molecules and their binding protein, follistatin, are widely expressed, and their production is increased in many acute and chronic inflammatory conditions. Synthesis and release of the activins are stimulated by inflammatory cytokines, Toll-like receptor ligands, and oxidative stress. The activins interact with heterodimeric serine/threonine kinase receptor complexes to activate SMAD transcription factors and the MAP kinase signaling pathways, which mediate inflammation, stress, and immunity. Follistatin binds to the activins with high affinity, thereby obstructing the activin receptor binding site, and targets them to cell surface proteoglycans and lysosomal degradation. Studies on transgenic mice and those with gene knockouts, together with blocking studies using exogenous follistatin, have established that activin A plays critical roles in the onset of cachexia, acute and chronic inflammatory responses such as septicemia, colitis and asthma, and fibrosis. However, activin A also directs the development of monocyte/macrophages, myeloid dendritic cells, and T cell subsets to promote type 2 and regulatory immune responses. The ability of both endogenous and exogenous follistatin to block the proinflammatory and profibrotic actions of activin A has led to interest in this binding protein as a potential therapeutic for limiting the severity of disease and to improve subsequent damage associated with inflammation and fibrosis. However, the ability of activin A to sculpt the subsequent immune response as well means that the full range of effects that might arise from blocking activin bioactivity will need to be considered in any therapeutic applications.

The roles of activin A and its binding protein, follistatin, in inflammation and tissue repair.

Activin A, a member of the transforming growth factor-β superfamily of cytokines, is a critical controller of inflammation, immunity and fibrosis. It is rapidly released into the blood following a lipopolysaccharide challenge in experimental animals, through activation of the Toll-like receptor 4 signalling pathway. Blocking activin action by pre-treatment with its binding protein, follistatin, modifies the inflammatory cytokine cascade, and reduces the severity of the subsequent inflammatory response and mortality. Likewise, high serum levels of activin A are predictive of death in patients with septicaemia. However, activin A has complex immunomodulatory actions. It is produced by inflammatory macrophages, but can regulate either pro- or anti-inflammatory responses in these cells, depending on their prior activation status. Activin A is also produced by Th2 cells, and stimulates antibody production by B cells and the development of regulatory T cells. Production of activin A during inflammatory responses stimulates fibrosis and tissue remodelling, and follistatin inhibits these actions of activin A. The modulation of activin by follistatin may represent an important therapeutic target for the modulation and amelioration of inflammatory and fibrotic disorders.