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M P Hedger

Publications and source records attributed to M P Hedger.

At least 19 recordsLinked to original sources
PubMed2002

Expression of monocyte chemoattractant protein-1 and macrophage colony-stimulating factor in normal and inflamed rat testis.

Macrophages are numerous in the testicular interstitial tissue under normal conditions and increase during inflammation. The mechanisms involved are poorly characterized. Expression of the macrophage-regulating cytokines monocyte chemoattractant protein (MCP)-1 and macrophage colony-stimulating factor (M-CSF) was examined in the adult rat testis before and after an i.p. injection of an inflammatory stimulus, lipopolysaccharide (LPS). In the normal testis, M-CSF was readily observed using Northern blot and Western blot analysis. In contrast, MCP-1 was not detectable by Northern blot in the normal testis, but was detected using RT-PCR amplification and a sensitive ELISA. After LPS treatment, testicular MCP-1 mRNA and protein expression increased dramatically (up to 400-fold). In-situ hybridization for MCP-1 revealed that production was confined to the interstitium of the inflamed testis, in Leydig cells, peritubular cells, perivascular cells and monocyte-like macrophages, but not in tissue-resident macrophages. Unlike MCP-1, M-CSF mRNA and protein expression in the testis increased only marginally, if at all, after LPS treatment. These results suggest that MCP-1 stimulates the increase in intratesticular macrophages that accompanies LPS-induced inflammation in vivo. Together with M-CSF, MCP-1 may also play a role in maintaining the resident macrophage population of the normal testis.

Animals↗
PubMed2001

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.

Activins↗
PubMed2001

Differential effects of dexamethasone treatment on lipopolysaccharide-induced testicular inflammation and reproductive hormone inhibition in adult rats.

A single intraperitoneal injection of lipopolysaccharide (LPS) causes a biphasic suppression of testicular steroidogenesis in adult rats, with inhibition at 6 h and 18-24 h after injection. The inhibition of steroidogenesis is independent of the reduction in circulating LH that also occurs after LPS treatment, indicating a direct effect of inflammation at the Leydig cell level. The relative contributions to this inhibition by intratesticular versus systemic responses to inflammation, including the adrenal glucocorticoids, was investigated in this study. Adult male Wistar rats (eight/group) received injections of LPS (0.1 mg/kg i.p.), dexamethasone (DEX; 50 microg/kg i.p.), LPS and DEX, or saline only (controls), and were killed 6 h, 18 h and 72 h later. Treatment with LPS stimulated body temperature and serum corticosterone levels measured 6 h later. Administration of DEX had no effect on body temperature, but suppressed serum corticosterone levels. At the dose used in this study, DEX alone had no effect on serum LH or testosterone at any time-point. Expression of mRNA for interleukin-1beta (IL-1beta), the principal inflammatory cytokine, was increased in both testis and liver of LPS-treated rats. Serum LH and testosterone levels were considerably reduced at 6 h and 18 h after LPS treatment, and had not completely recovered by 72 h. At 6 h after injection, DEX inhibited basal IL-1beta expression and the LPS-induced increase of IL-1beta mRNA levels in the liver, but had no effect on IL-1beta in the testis. The effects of DEX on IL-1beta levels in the liver were no longer evident by 18 h. In LPS-treated rats, DEX caused a significant reversal of the inhibition of serum LH and testosterone at 18 h, although not at 6 h or 72 h. Accordingly, DEX inhibited the systemic inflammatory response, but had no direct effect on either testicular steroidogenesis or intra-testicular inflammation, at the dose employed. These data suggest that the inhibition of Leydig cell steroidogenesis at 6 h after LPS injection, which was not prevented by co-administration of DEX, is most likely due to direct actions of LPS at the testicular level. In contrast, the later Leydig cell inhibition (at 18 h) may be attributable to extra-testicular effects of LPS, such as increased circulating inflammatory mediators or the release of endogenous glucocorticoids, that were inhibited by DEX treatment. These data indicate that the early and late phases of Leydig cell inhibition following LPS administration are due to separate mechanisms.

Analysis of Variance↗
PubMed2000

Divergent cell-specific effects of activin-A on thymocyte proliferation stimulated by phytohemagglutinin, and interleukin 1beta or interleukin 6 in vitro.

Activin-A is a member of the transforming growth factor-beta (TGF-beta) cytokine family. Based on studies in several cell systems, activin-A has been postulated to be a specific inhibitor of the actions of the inflammatory cytokine, interleukin 6. In cultures of adult rat thymocytes, activin-A inhibited sub-optimal phytohemagglutinin-induced and interleukin 1beta-stimulated proliferation, as measured by [(3)H]-thymidine incorporation in vitro. In contrast with TGF-beta1, which exerted similar inhibitory effects on thymocyte proliferation, activin-A activity was reduced by increasing the concentration of phytohemagglutinin or addition of the reducing agent, beta-mercaptoethanol. Both activin-A and TGF-beta1 inhibited the in vitro production of interleukin 6 by thymocytes in the presence of phytohemagglutinin and interleukin 1beta. In the presence of exogenous interleukin 6, however, both activin-A and TGF-beta1 stimulated thymocyte proliferation. These data suggest that activin-A inhibits thymocyte growth and differentiation, at least in part, by inhibiting endogenous production of interleukin 6, but stimulates thymocyte growth when exogenous interleukin 6 is present in vitro. These data indicate that activin interacts with other cytokines to exert complex regulation of T cell development, and is not an inhibitor of interleukin 6 action in all cell systems.

Activins↗
PubMed2000

Inducible nitric oxide synthase in the rat testis: evidence for potential roles in both normal function and inflammation-mediated infertility.

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.

Animals↗
PubMed2000

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, and subsequently apoptosis of spermatocytes at stages II-V. These data indicate that mild inflammation causes local inhibition of Leydig cell function with relatively little spermatogenic damage. The pathological changes in spermatogenic function during severe inflammation are most likely due to direct effects of inflammatory mediators on the seminiferous epithelium or testicular vasculature, rather than inhibition of the brain-pituitary-Leydig cell axis.

Animals↗
PubMed2000

Cloning and regulation of the rat activin betaE subunit.

Using a combination of polymerase chain reaction (PCR) procedures, we have cloned and sequenced the rat activin beta(E) subunit cDNA. The putative protein corresponding to the prepro-activin beta(E) subunit was predicted to comprise 350 amino acids which, when cleaved between amino acid residues 236 and 237, would yield a mature polypeptide of approximately M(r) 12 500 with a predicted pI of 5.1. Two cDNA transcripts for activin beta(E) were identified; these differed by 738 bp in the 3'-untranslated region. Activin beta(E) mRNA transcripts were expressed only in rat liver and lung tissue as assessed by Northern blotting and PCR analysis. Relatively higher levels of both transcripts were found in the liver, whereas the lung contained lower levels that were detectable by PCR only. In situ hybridisation data showed that, within the liver, activin beta(E) mRNA was localised to hepatocytes. In vivo treatment with lipopolysaccharide as a means of activating the immune system and the hepatic acute-phase response resulted in stimulated activin beta(E) mRNA levels, compared with untreated, control rats. This increased expression was accompanied by a preferential increase in the amount of the long activin beta(E) transcript over the shorter transcript. These findings suggested that the two activin beta(E) mRNA transcripts may be products of alternative splicing events or use alternative polyadenylation sites which are differentially regulated during inflammation. These data provide evidence of a role for activin beta(E) in liver function and inflammation in the rat.

Activins↗
PubMed1999

Activin A regulates growth and acute phase proteins in the human liver cell line, HepG2.

Activin, and its binding protein, follistatin, are up-regulated by mediators of inflammation, and recent studies have demonstrated that activin A can block the activity of the key inflammatory cytokine, interleukin-6 (IL-6). These findings thereby implicate activin and follistatin in the control of the inflammatory cascade. In this study, interactions between interleukin-1beta (IL-1beta), IL-6 and activin were examined the human liver cell line, HepG2, for their effect on cell proliferation and the production of the acute phase proteins, haptoglobin and alpha1-acid glycoprotein (alpha1-AGP). IL-1beta and activin A, but not IL-6, inhibited the proliferation of HepG2 cells. Activin A together with IL-1beta caused a greater inhibition of proliferation than either factor alone, and the inhibitory effects of activin A were blocked by the addition of follistatin to the cultures. Activin A alone inhibited the production of haptoglobin but did not affect alpha1-AGP concentrations. However, activin A suppressed the stimulatory effects of IL-6 on the production of both haptoglobin and alpha1-AGP. Production of follistatin by HepG2 cells was stimulated by activin A, but was inhibited by both IL-1beta and IL-6, indicating a complex regulatory loop is operable to modulate the effects of activin A during inflammation. Taken together, these data suggest that activin A interacts with IL-1beta and IL-6 to regulate and coordinate the production of acute phase proteins during an inflammatory episode.

Activins↗
PubMed1999

A switch in the cellular localization of macrophage migration inhibitory factor in the rat testis after ethane dimethane sulfonate treatment.

Macrophage migration inhibitory factor (MIF), one of the first cytokines to be discovered, has recently been localized to the Leydig cells in adult rat testes. In the following study, the response of MIF to Leydig cell ablation by the Leydig cell-specific toxin ethane dimethane sulfonate (EDS) was examined in adult male rats. Testicular MIF mRNA and protein in testicular interstitial fluid measured by ELISA and western blot were only marginally reduced by EDS treatment, in spite of the fact that the Leydig cells were completely destroyed within 7 days. Immunohistochemistry using an affinity-purified anti-mouse MIF antibody localized MIF exclusively to the Leydig cells in control testes. At 7 days post-EDS treatment, there were no MIF immunopositive Leydig cells in the interstitium, although distinct MIF immunostaining was observed in the seminiferous tubules, principally in Sertoli cells and residual cytoplasm, and some spermatogonia. A few peritubular and perivascular cells were also labelled at this time, which possibly represented mesenchymal Leydig cell precursors. At 14 and 21 days, Sertoli cell MIF immunoreactivity was observed in only a few tubule cross-sections, while some peritubular and perivascular mesenchymal cells and the re-populating immature Leydig cells were intensely labeled. At 28 days after EDS-treatment, the MIF immunostaining pattern was identical to that of untreated and control testes. The switch in the compartmentalization of MIF protein at 7 days after EDS-treatment was confirmed by western blot analysis of interstitial tissue and seminiferous tubules separated by mechanical dissection. These data establish that Leydig cell-depleted testes continue to produce MIF, and suggest the existence of a mechanism of compensatory cytokine production involving the Sertoli cells. This represents the first demonstration of a hitherto unsuspected pattern of cellular interaction between the Leydig cells and the seminiferous tubules which is consistent with an essential role for MIF in male testicular function.

Animals↗
PubMed1999

Mitosis of resident macrophages in the adult rat testis.

Resident macrophages are maintained at a comparatively high, yet stable, tissue concentration in the adult rat testis. After destruction of Leydig cells by ethane dimethane sulphonate treatment, the number of resident macrophages increases briefly and then decreases to below normal values, but returns to normal after the reappearance of Leydig cells. The mechanisms by which the adult testicular macrophage population is maintained, either by monocyte recruitment or by mitosis of the resident macrophages, have not been examined. An immunohistochemical dual labelling approach using a specific monoclonal antibody for resident macrophages, ED2, and markers of mitotic activity (bromodeoxyuridine incorporation and expression of the proliferating cell nuclear antigen) was used to investigate resident macrophage proliferation in Bouin's-fixed paraffin wax-embedded adult rat testes. Detection of the normally fixation sensitive antigen recognized by ED2 was achieved by using a decreased fixation time and antigen retrieval. Peaks of resident macrophage mitotic activity were observed during the phases of macrophage proliferation immediately after ethane dimethane sulphonate treatment and during the recovery phase associated with Leydig cell restoration. These data demonstrate that resident macrophages have the capacity to proliferate within the adult rat testis and, thus, this population of resident macrophages is maintained, at least in part, by mitotic division in situ.

Analysis of Variance↗
PubMed1999

A sensitive and specific in vitro bioassay for activin using a mouse plasmacytoma cell line, MPC-11.

A new in vitro bioassay for activin was developed using the mouse plasmacytoma cell line, MPC-11. Human recombinant (hr) activin A dose-dependently inhibited the proliferation of these cells, whereas a range of other factors, including inhibin, follistatin and transforming growth factor-beta1, -beta2 and -beta3 had no effect. Conditioned medium containing activin B induced an inhibition similar to hr-activin A. The inhibitory influence of activin A could be blocked by follistatin, but not by hr-inhibin A. This bioassay had a sensitivity for activin A of around 0.4 ng/ml, an ED50 response of 3.5 ng/ml, and an intra-assay coefficient of variation of <11%. It offers substantial advantages over existing in vitro activin bioassays in terms of ease of use, specificity and throughput. The utility of the MPC-11 bioassay was demonstrated in the purification of activin from amniotic fluid, where an almost identical profile of bioactive activin A was detected compared with the pituitary cell bioassay of activin. Bioactive activin could also be detected in unpurified ovine allantoic and amniotic fluids and bovine follicular fluid. Measuring activin in untreated and heat-treated human sera or seminal plasma was hampered by a non-specific inhibitory effect, so that several serum samples did not run parallel with the hr-activin A standard. This inhibitory effect by serum could not be overcome by addition of follistatin, suggesting it is not activin-like bioactivity. This new bioassay for activin demonstrates widespread applicability for monitoring of purified or partially purified samples during purification procedures, bioactivity measurements, receptor-binding studies and assays of cell culture medium.

Activins↗
PubMed1998

Immunoregulatory activity in adult rat testicular interstitial fluid: roles of interleukin-1 and transforming growth factor beta.

Studies on the effect of rat testicular interstitial fluid (IF) on T-cell function have reported both stimulatory and inhibitory actions. Specific cytokines produced within the testis, particularly interleukin-1 (IL-1) and transforming growth factor beta (TGFbeta), may contribute to these apparently conflicting observations. In proliferation assays employing lectin- or antibody-activated thymocytes or mature T cells in vitro, adult rat testicular IF stimulated T-cell activation and/or proliferation at low assay doses and was inhibitory at higher doses. The stimulatory activity was blocked by recombinant IL-1 receptor antagonist. The inhibitory activity was not affected by a polyspecific TGFbeta antiserum. The biological characteristics of the inhibitor were distinct from those of a similar, but considerably less potent, activity in platelet-depleted serum. These data demonstrate that rat testicular IF contains biologically significant concentrations of IL-1 but has a predominantly inhibitory action on T-cell responses. The factor predominantly responsible for this inhibitory activity displays a relatively large apparent molecular weight, is protease sensitive and partially heat labile, but does not appear to be one of the known mammalian TGFbeta isoforms.

Animals↗
PubMed1998

Immunoregulatory activity in adult rat testicular interstitial fluid: relationship with intratesticular CD8+ lymphocytes following treatment with ethane dimethane sulfonate and testosterone implants.

Regulation of T-cell traffic and function in the adult rat testis was assessed following treatment with the specific Leydig cell cytoxin, ethane dimethane sulfonate (EDS), and s.c. testosterone implants to prevent Leydig cell recovery. The distribution of T-cell subsets in the testis was determined immunohistochemically using stereological techniques. Testicular T cell-inhibiting activity in the interstitial fluid was measured using a phytohemagglutinin-activated rat thymocyte proliferation bioassay. The mostly cytotoxic CD8+ T-cell subset predominated over the CD4+ (regulatory) T-cell subset in the normal rat testis. Destruction of the Leydig cells caused a rapid preferential increase in testicular CD4+ T cells, which was followed by an increase in both the CD8+ subset and T cell-inhibiting activity in the Leydig cell-deficient testis. After Leydig cell recovery, there was a significant shift toward the CD8+ T-cell subset in the EDS-treated testis but not in the EDS-treated/testosterone-implanted testis. Total T-cell numbers and inhibitory activity in the testis returned to control levels regardless of whether the Leydig cells were allowed to recover. The level of inhibitory activity was closely related to the number of CD8+ T cells in the testis across all experimental groups, but it showed no relationship with pituitary hormones, macrophage numbers, or intratesticular testosterone levels. The data suggest that 1) cytotoxic lymphocytes have a potentially significant role in testicular function and 2) T cell-inhibiting activity in the testis interstitium is not substantially affected by changes in pituitary hormones or Leydig cell function, but appears to be related to local changes in immune activity.

Animals↗
PubMed1998

Characterization of lymphocytes in the adult rat testis by flow cytometry: effects of activin and transforming growth factor beta on lymphocyte subsets in vitro.

The rat testis is considered to be an immunologically privileged site because of its reduced capacity to support antigen-specific immune responses. To understand this phenomenon, it is essential to characterize both the lymphocyte subpopulations normally present in the testis and their regulation by testicular cytokines. Peripheral blood was obtained from adult male Dark Agouti or Sprague-Dawley rats, and testicular interstitial tissue was collected after perfusion of the testes to remove blood. Blood and testis lymphocytes were isolated using discontinuous Percoll density gradients, and the testicular lymphocytes were further purified by selective adherence to remove mononuclear phagocytes. The isolated lymphocytes were analyzed by flow cytometry using specific monoclonal antibodies and fluorescein labeling and were enumerated as total T cells, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells. In contrast to peripheral blood, in which the CD4+ T-cell subset was the major lymphocyte subset, rat testis T cells were predominantly of the CD8+ subset, and a large population of NK cells also were present. Subsequently, peripheral blood lymphocytes were stimulated with the polyclonal T-cell activator, phytohemagglutinin, and cultured in the presence of activin, inhibin, or transforming growth factor beta (TGFbeta) prior to flow cytometric analysis. Activin and TGFbeta suppressed T-cell proliferation without any selective effect on either T-cell subset, and inhibin had no effect. The predominance of CD8+ T cells and NK cells, and the relatively minor proportion of CD4+ T cells, are consistent with both increased cellular immune surveillance and a reduced capacity for initiating antigen-specific immune responses in the adult rat testis.

Activins↗
PubMed1997

Comparison of LHRH-peptidase and plasminogen activator activity in rat testis extracts.

Testicular LHRH-peptidase and testicular urokinase-type plasminogen activator are Sertoli cell-secreted proteases which display similar molecular properties. However, there is relatively little information regarding the substrate specificity and potential cross-reactivity of these enzymes. Testicular extracts were prepared from homogenates of whole rat testes and assessed by LHRH-peptidase assay, and by radial caseinolysis assays for plasminogen activator and plasmin-like activity. Following partial purification of the protease activities in testicular extracts by gel filtration and ion-exchange chromatography, it was confirmed that testicular LHRH-peptidase and plasminogen activator are clearly separable. There was no detectable plasmin-like activity in the testicular extracts; however, the extracts were found to contain an inhibitor, or inhibitors, of both plasminogen activator and plasmin activity. In addition to LHRH and Gly6-substituted LHRH analogues, the partially purified LHRH-peptidase degraded both angiotensins I and II, but not the gonadotrophin-releasing-hormone-associated peptide derived from the LHRH precursor molecule. These properties of the LHRH-peptidase provide further evidence that it is a testis-specific prolyl endopeptidase, involved in regulating and/or limiting peptide activity in the testis.

Animals↗