We undertook the present studies to determine if clodronate-containing liposomes have direct effects on Leydig cells. Macrophages and Leydig cells were isolated and maintained separately in culture. Following treatment with clodronate-containing liposomes, macrophages were killed in a dose-response fashion over a range of 5-200 microliters liposomes. By comparison, a 500 microliters dose was required to kill Leydig cells, but this was not dependent upon clodronate since liposomes containing buffer elicited an identical response. The concentration of testosterone in medium from Leydig cells treated with clodronate-containing liposomes was significantly reduced compared with untreated cells. However, we subsequently found that liposomes can adsorb testosterone. Therefore, testosterone production was determined at various times following removal of liposomes from Leydig cells, thereby circumventing this complication. It was found that testosterone production was not altered by liposomes under these conditions. Finally, free clodronate had no effect on testosterone production, even at doses representing the amount present within the 500 microliters dose of liposomes. In summary, clodronate-containing liposomes killed testicular macrophages at a far smaller dose than required to kill Leydig cells. Most importantly, neither liposomes no free clodronate had a direct effect on testosterone production. Thus, clodronate-containing liposomes represent a valuable tool to study Leydig cell-macrophage interactions.

SUMMARY

Adult female rats undergoing their first lactation were experimentally weaned by removing their pups on day 4 of lactation. The macrophages of the mammary gland were labelled with trypan blue or horseradish peroxidase. Treatment with trypan blue showed the macrophages to be present in the connective tissue stroma from the 4th day of lactation to the 5th day of involution. However, on day 6 of involution they were congregated around the remaining islets of alveoli. Other cells which took up the dye were found in the epithelium of the alveoli. They were few in number on day 4 of lactation, increased numerically during the first 2 days of involution, and decreased again by day 6 of involution. Since they took up the dye in a similar manner to the lymphocytes of the regional lymph node, it is believed that they were lymphocytes. Treatment with ICI 46,474 in no way affected the course of mammary involution up to day 6. Treatment with horseradish peroxidase on day 4 of lactation or day 2 of involution resulted in labelling of the macrophages, which were found scattered in the stroma. However, by the 4th and 6th day of involution the macrophages were found to a greater extent in and around the remaining islets of alveoli.

ABSTRACT

The objective of this study was to examine the role of intertubular macrophages in modifying the response of the rat testis to stimulation by human chorionic gonadotrophin (hCG). The phagocytic activity of macrophages was stimulated by a unilateral intratesticular injection of polystyrene latex beads. Latex beads were engulfed by the resident macrophages and retained within their cytoplasm. Contralateral testes received injection of vehicle alone. A group of control rats was killed 3 days later; other groups received 100 IU hCG s.c. and the morphological and functional responses of the testes were examined 12, 24 and 48 h later. Spermatogenesis was unaffected in control rats, whereas in the testes of all hCG-treated rats leukocytes infiltrated into the intertubular tissue and the seminiferous tubules exhibited focal disruptions of spermatogenesis which were more severe in testes containing activated macrophages. Spermatogenic disruption was dependent upon the stage of the spermatogenic cycle, with the maximum tubule degeneration occurring at or near stages III and IX–XI. However these changes were not a consequence of androgen deprivation, since no consistent correlation was demonstrated between alterations in testosterone levels in testicular interstitial fluid and the accompanying damage to germ cells. It is concluded that hCG alone or in combination with activated macrophages induces an inflammatory-type response of the intertubular tissue and localized degeneration of the seminiferous epithelium. The antispermatogenic effects of hCG may have important implications for in-vivo investigations of Leydig cell function in laboratory animals and for the efficacy of hCG administration used in the clinical treatment of male hypogonadism.

Journal of Endocrinology (1989) 121, 285–292

The purpose of this investigation was to study the mechanism of action of a macrophage-derived factor that stimulates steroid production by Leydig cells. This factor increased testosterone production within 30 min, and reached a half-maximal response by 6-8 h. At a maximal dose, it stimulated testosterone production 20-fold at 24 h. Its efficacy was consistently higher than that achieved with a maximal dose of human chorionic gonadotropin (hCG). However, Leydig cells treated with a maximal dose of both the macrophage-derived factor and hCG secreted the same amount of testosterone as when given a maximal dose of only the macrophage-derived factor. The macrophage-derived factor did not require new protein synthesis to stimulate testosterone production, nor did it alter the amount of steroidogenic acute regulatory protein (StAR). While the macrophage-derived factor required an active cholesterol side-chain cleavage complex system, it did not alter the capacity of this enzyme complex. Finally, the macrophage-derived factor was unable to stimulate the production of progesterone by isolated mitochondria. In summary, the macrophage-derived factor is a highly active, acute regulator of steroidogenesis that acts through a high capacity StAR-independent pathway.

Oxidized low density lipoproteins (LDL) are highly suspected of initiating the atherosclerosis process. Thyroid hormones and structural analogues have been reported to protect LDL from lipid peroxidation induced by Cu2+ or the free radical generator 2,2'-azobis-'2-amidinopropane' dihydrochloride in vitro. We have examined the effects of thyroid compounds on macrophage-induced LDL oxidation. Human monocyte-derived macrophages (differentiated U937 cells) were incubated for 24 h with LDL and different concentrations (0-20 microM) of 3,5,3'-triiodo-l -thyronine (T3), 3,5,3',5'-tetraiodo-L-thyronine (T4), 3,3',5'-tri-iodo-l -thyronine (rT3), the T3 acetic derivative (3,5,3'-tri-iodothyroacetic acid; TA3) or L-thyronine (T0) (experiment 1). Cells were also preincubated for 24 h with 1 or 10 microM of the compounds, washed twice, then incubated again for 24 h with LDL (experiment 2). Oxidation was evaluated by measurement of thiobarbituric acid-reactive substances (TBARS) and cell viability by lactate deshydrogenase release. In experiment 1, T0 had no effect, whereas the other compounds decreased LDL TBARS production, but T3 and TA3 were less active than T4 and rT3 (IC50: 11.0 +/- 2.6 and 8.1 +/- 0.8 vs 1.4 +/- 0.5 and 0.9 +/- 0.3 microM respectively). In experiment 2, the compounds at 1 microM had no effect; at 10 microM, T3 and rT3 slightly reduced LDL TBARS production, whereas TA3 and T4 inhibited it by about 50% and 70% respectively. TBARS released by the cells were also highly decreased by T3, T4, rT3 and TA3 in experiment 1, but only by T3 (30%) and T4 (70%) in experiment 2. Cell viability was not affected by the compounds except slightly by TA3 at 10 microM. The data suggested that the physico-chemical antioxidant capacity of thyroid compounds was modulated by their action on the intracellular redox systems of macrophage. Overall cellular effects of T3 led to a reduction of its antioxidant capacity whereas those of T4 increased it. Thus T4 might protect LDL against cellular oxidation in vivo more than T3.

Macrophage migration inhibitory factor (MIF) is an essential regulator of the macrophage responses to endotoxin. MIF also has the ability to override the anti-inflammatory actions of glucocorticoids during an immune response, and is thus an important pro-inflammatory factor. The presence of MIF in cells of the anterior pituitary has been described, and high levels of MIF in other rapidly proliferating tIssues have also been demonstrated. It has been hypothesised that MIF release from these cells is influenced by the hypothalamo-pituitary-adrenal axis, and that ACTH and MIF are released simultaneously to exert counter-regulatory effects on cortisol. However, another intracellular role for MIF has also been suggested as it has been shown that MIF exerts an effect on the inhibitory cell cycle control protein p27 through an interaction with Jab1, a protein implicated in p27 degradation. We studied MIF expression in different normal and adenomatous human pituitary samples using immunohistochemistry and RT-PCR. There was evidence of co-immunoprecipitation of MIF with Jab1, suggesting an interaction of the two proteins. Our results showed that there is increased expression of MIF protein in the nuclei of all pituitary adenomas compared with normal tIssue (P=0.0067), but there was no statistically significant difference in nuclear MIF expression between the different adenoma types. Nuclear MIF expression correlated positively with p27 and its phosphorylated form in normal tIssue (P=0.0028 and P<0.0001); however, this relationship was not seen in the adenoma samples. Cytoplasmic expression of MIF was found to be variable both in normal and adenomatous samples, with no consistent pattern. MIF mRNA was demonstrated to be present in all tumour and normal samples studied. Somatotroph tumours showed higher MIF mRNA expression compared with normal pituitary or other types of adenomas. In conclusion, MIF is expressed in cell nuclei in pituitary adenomas to a greater extent than in normal pituitary tIssue. We speculate that it may play a role in the control of the cell cycle, but whether its higher level in adenomas is a cause or a consequence of the tumorigenic process remains to be clarified.

Abstract

Macrophages are a common cell type in the testicular interstitium of the rat and are morphologically and functionally related to Leydig cells. We investigated the number of macrophages and Leydig cells in long-term (24 weeks) hypophysectomized (LTHX) or sham-operated rats. LTHX rats showed a 76% decrease in the number of macrophages, whereas the number of Leydig cells was only slightly decreased (by 18%). The profile areas of both macrophages and Leydig cells were very much decreased (46% and 66% respectively).

Sham-operated and LTHX rats were treated with vehicle or human FSH and LH (hFSH/hLH; 75 IU/kg body weight per day) for 1 week. This treatment induced a 286% increase in the number of macrophages and a 32% increase in the number of Leydig cells in LTHX rats. The profile areas of macrophages and Leydig cells were also increased (212% and 184% respectively). About 80% of macrophages showed vacuolization of the cytoplasm. Gonadotrophin treatment did not induce changes in cell numbers in sham-operated animals but about 30% of macrophages showed large cytoplasmic vacuoles.

Vehicle- or hormone-treated LTHX rats were given a single injection of ethylene dimethane sulphonate (EDS) and killed 72 h later. Leydig cells were absent from the testicular interstitium of sham-operated rats but there were large numbers of dead Leydig cells (about 40% of the pre-existing population) in the testicular interstitium of LTHX rats 3 days after EDS treatment. Complete clearance of the testicular interstitium from EDS-killed Leydig cells was found in LTHX rats treated with hFSH/hLH. These results indicate that the decreased number and size and the defective function of testicular macrophages in LTHX rats can be restored by treatment with gonadotrophins.

Journal of Endocrinology (1994) 140, 399–407

ABSTRACT

It has been suggested that the effects of dysthyroidism on resident immunocompetent cells of the extraocular muscles may play a role in the pathogenesis of Graves' ophthalmopathy. The distribution of such cells was therefore studied in extraocular muscles of rats that were made hyper- or hypothyroid by the oral administration of thyroxine or propylthiouracil respectively. Skeletal muscles were studied for comparison. The cell distributions were analysed in cryostat cross-sections subjected to a two-step immunoperoxidase method using well-characterized monoclonal antibodies against T cells, B cells, macrophages and MHC class II antigens.

The extraocular muscles of control (euthyroid) rats contained numerous macrophages, fewer MHC-II positive cells and T cells and no B cells. Differences in the distribution of immunocompetent cells were found in control rats, between skeletal and extraocular muscles as well as within the various recti eye muscles. This particular tissue distribution resembles that previously reported for human extraocular and skeletal muscles.

Quantitative analysis showed that experimental dysthyroidism only affected cell populations in the extraocular muscles. Significant effects on the number of macrophages were observed in the inferior rectus muscle of both hypo- and hyperthyroid rats, this was most pronounced in the orbital layer of the muscles. Both hyper- and hypothyroidism appear to affect local cell distributions in a tissue-specific manner. The presently observed site-dependent effects of dysthyroidism on local immunocompetent cell populations may have relevance for the differential involvement of muscular tissues in Graves' ophthalmopathy.

Journal of Endocrinology (1992) 135, 485–493

The immunological and neuroendocrine properties of macrophage migration inhibitory factor (MIF) are diverse. In this article we review the known cellular, molecular and genetic properties of MIF that place it as a key regulatory cytokine, acting within both the innate and adaptive immune responses.The unexpected and paradoxical induction of MIF secretion by low concentrations of glucocorticoids is explored. The role of MIF as a locally acting modulator of glucocorticoid sensitivity within foci of inflammation is also discussed. MIF has no homology with any other pro-inflammatory cytokine and until recently lacked a recognised transmembrane receptor. MIF has also been shown to be directly taken up into target cells and to interact with intracellular signalling molecules, including the Jun activation domain-binding protein Jab-1.Comprehensive analysis of the MIF gene has identified important functional polymorphisms and a series of genetic studies has revealed both association and linkage of MIF with inflammatory diseases. Altered MIF regulation may therefore be pivotal to acquiring chronic inflammation following an innate immune response.

Glucocorticoids represent one of the most effective clinical treatments for a range of inflammatory conditions, including severe acute inflammation. Although glucocorticoids are known to affect processes involved in the initiation of inflammation, the influence of glucocorticoids on the mechanisms by which acute inflammation normally resolves have received less attention. Apoptosis of granulocytes present at inflamed sites leads to their rapid recognition and internalisation by macrophages, a process which may be important for resolution of inflammation. However, if clearance of either eosinophils or neutrophils is impaired, these cells rapidly undergo secondary necrosis leading to release of pro-inflammatory mediators from the phagocyte, potentially prolonging inflammatory responses. Physiologically relevant concentrations of glucocorticoids accelerate eosinophil apoptosis whilst delaying neutrophil apoptosis during in vitro culture. Here we discuss key pathways regulating the granulocyte apoptotic programme and summarise the effects of glucocorticoids on monocyte differentiation and the consequent changes to apoptotic cell clearance capacity. Definition of the mechanisms underlying resolution of inflammatory responses following glucocorticoid treatment may unveil new targets for modulation of inflammatory disease, allowing co-ordinated augmentation of granulocyte apoptosis together with increased macrophage capacity for clearance of apoptotic cells.