Tissue-specific pretranslational regulation of complement production in human mononuclear phagocytes

The net production of the complement protein C2, C4, and factor B differ among mononuclear phagocytes from peripheral blood and different tissues in experimental animals and in humans. To examine the mechanisms that regulate these differences in humans, the proportions of C2-producing cells, the average single-cell production rate of C2, the posttranslational glycosylation and kinetics of secretion of C2 and factor B, and the amounts of C2 and factor B mRNA were examined in freshly isolated peripheral blood monocytes, monocytes maintained up to 2 wk in culture, and freshly isolated tissue macrophages from breast milk and bronchoalveolar lavage. In addition, the biosynthesis of two other proteins synthesized and secreted by mononuclear phagocytes, C3 and lysozyme, were examined. We report that despite comparable rates of C3 and lysozyme synthesis and similar processing and kinetics of secretion of C2 and factor B, the freshly isolated tissue macrophage differs from the monocyte-derived macrophage in the proportion of C2-producing cells, in the average single-cell production rate of complement, and in the amounts of specific C2 and factor B mRNA. These differences are tissue specific, because C2-specific mRNA content in bronchoalveolar macrophages is considerably greater than in breast milk macrophages, although the amounts of factor B mRNA are comparable. These data suggest that tissue-specific regulation of complement production in human mononuclear phagocytes occurs at a pretranslational level. These studies now provide a basis for investigation of the molecular effects of agents that modulate the biologic functions of monocytes and macrophages.     

A1 and A2 adenosine receptor regulation of erythropoietin production

The effects of adenosine (ADE) and ADE agonists on erythropoietin (Ep) production were determined using percent (%) 59Fe incorporation in red cells of exhypoxic polycythemic mice. The hemisulfate salt of ADE produced a significant increase in % 59Fe incorporation in response to hypoxia in concentrations of 400 to 1600 nmol/kg/day (i.v.). 5'-N-ethyl-carboxamideadenosine (NECA), a selective A2 receptor agonist, increased radioiron incorporation in a dose-dependent manner (10-100 nmol/kg/day, i.v.). In contrast, N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, did not affect radioiron incorporation in concentrations up to 1600 nmol/kg/day (i.v.). Albuterol, a beta 2-adrenergic agonist, enhanced % 59Fe incorporation in polycythemic mice and low doses of CHA (50 and 100 nmol/kg/day), which were not effective alone on % 59Fe incorporation in polycythemic mice exposed to hypoxia, inhibited the enhancement in radioiron induced by albuterol (25 and 100 micrograms/kg/day, i.p.) plus hypoxia. Theophylline (20 and 80 mg/kg/day, i.p.), a well-known antagonist of ADE receptors, blocked the ADE and NECA enhancement in radioiron incorporation at a dose of theophylline alone which produced only a slight enhancement of % 59Fe incorporation. These results suggest that ADE may both inhibit through A1 receptor activation and increase via A2 receptor stimulation the production of Ep.     

Ultrastructure of cyclic changes in the murine uterus,cervix, and vagina

The regional and cyclic changes in the murine genital epithelium were studied by transmission and scanning electron microscopy to provide a morphological standard to serve as a basis for investigation of host-parasite relationships in genital infections. Thus, we examined not only mucosal epithelial cell changes, but also surface mucus, normal flora and inflammatory cells. Ultrastructurally, at proestrus/estrus, we found uterine and most cervical epithelial cells covered with microvilli overlaid with mucus-like secretions and evidence of internal secretory activity. There was little normal flora anywhere in the tract. At early metestrus, we found squamous cervicovaginal epithelial cells with low discontinuous microrugae, extensive normal flora and many neutrophils beginning to migrate through the epithelium. The flora and neutrophils could explain the relative lack of susceptibility to infection at that time. At diestrus the appearance of a newly regenerated epithelium and lack of normal flora suggested that initiation of infection could occur at this stage; however, the presence of large numbers of neutrophils ready to phagocytize invading bacteria indicated a deterrent to infection. This study of cyclic changes in flora, mucus, neutrophils and epithelial cells provided ultrastructural evidence to support an earlier hypothesis that the greatest susceptibility to gonococcal infection in mice occurred at proestrus/estrus.     

Glycosylation of the murine erythropoietin receptor

Murine erythropoietin-responsive Rauscher Red 5-1.5 cells were used to determine the contribution of glycosylation to the size and function of the erythropoietin receptor. The half life of the receptors was determined to be 4 h. The number of receptors was not significantly decreased in cells treated for 48 h with inhibitors of glycosylation (tunicamycin, glucosamine or swainsonine) and their affinity was slightly enhanced in tunicamycin- or glucosamine-treated cells. Erythropoietin was cross-linked with two proteins of 104 and 86 kDa. Their molecular masses were not significantly reduced in cells treated with the glycosylation inhibitors. When immunoprecipitated cross-linked receptors were digested with endoglycosidases, the molecular masses of both proteins were only slightly modified giving values of 100 and 82 kDa. Thus we can conclude that the proteins cross-linked to erythropoietin are very weakly glycosylated.     

The role of prostaglandins in the production of erythropoietin (ESF) by the kidney. II. Effects of indomethacin on erythropoietin production following hypoxia in dogs

In order to determine the sequence of events leading to the increase in ESF production following renal hypoxia, the possible role of prostaglandins in the generation of ESF by the kidney was assessed. ESF production following exposure to hypoxia and indomethacin (I) treatment were studied in dogs. Plasma ESF titers were measured in carotid artery blood before the hypoxic stimulus, and after 1, 3 and 5 hours exposure. It was found that plasma ESF levels were significantly elevated in 6 dogs exposed to an hypoxic stimulus while anesthetized with pentobarbital by breathing an atmosphere of reduced oxygen (8% oxygen, 5% carbon dioxide and 87% nitrogen) for 3 and 5 hours. On the other hand, 6 dogs exposed to this hypoxic stimulus and pretreated with indomethacin (5 mg/kg orally 2 x), a drug which inhibits prostaglandins synthesis, did not show a significant increase in plasma ESF levels after exposure to hypoxia as compared to controls. These results indicate that renal prostaglandins play a role in ESF production after an hypoxic stimulus.     

In vivo and in vitro regulation of IgE production in murine hybridomas

Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.     

Unique patterns of androgen regulation of the expression of two genes in murine kidney

The kidneys of androgen stimulated mice exhibit a hypertrophic response but no hyperplasia or concomitant DNA replication. Androgens increase the expression of several genes in mouse kidney. The response of the beta-glucuronidase gene to testosterone in this tissue is characterized by a 1-2 day lag and relatively slow induction kinetics. The gene coding for kidney androgen-regulated protein (KAP) exhibits quite a different response to the hormone when compared on the basis of initial response to a given dose, dose required to produce maximal response, and apparent sensitivity to low levels of androgen-receptor complexes in renal nuclei. The analysis of the accumulation of the mRNAs produced by these two genes suggests that gene-specific differential sensitivity to androgen receptor complexes governs the development of the cellular male phenotype in this tissue.     

Mimicking the events of menstruation in the murine uterus

Menstruation and endometrial regeneration occur during every normal reproductive cycle in women and some Old World primates. Many of the cellular and molecular events of menstruation have been identified by correlative or in vitro studies, but the lack of a convenient model for menstruation in a laboratory animal has restricted functional studies. In this study, a mouse model for menstruation first described by Finn in the 1980s has been modified for use in a commonly used inbred strain of mouse. A decidual stimulus was applied into the uterine lumen of appropriately primed mice and leukocyte numbers and apoptosis were examined over time following progesterone withdrawal. Endometrial tissue breakdown was initiated after 12-16 h, and by 24 h, the entire decidual zone had been shed. Re-epithelialization was nearly complete by 36 h and the endometrium was fully restored by 48 h. Leukocyte numbers increased significantly in the basal zone by 12 h after progesterone withdrawal, preceding stromal destruction. Stromal apoptosis was detected by TUNEL staining at 0 and 12 h but decreased by 16 h after progesterone withdrawal. This mouse model thus mimics many of the events of human menstruation and has the potential to assist in elucidation of the functional roles of a variety of factors thought to be important in both menstruation and endometrial repair.     

Macrophages in murine uterus are immunosuppressive

The mechanisms by which the fetal allograft is protected from a maternal anti-fetal immune response are not understood. This study was designed to examine the possibility that tissues near the developing fetus contain immunoregulatory cells and to begin the process of identification of those cells. Dispersed uterine cell suspensions from pregnant Swiss/Webster mice consistently inhibited the responses of normal murine spleen cells to the polyclonal mitogen phytohemagglutinin (PHA). These suspensions contained few lymphocytes (mean 1%), but abundant macrophages (mean 28%), identified by morphology and Fcγ-receptor expression. Depletion of Fcγ-receptorpositive cells restored spleen cell (SC) responses to PHA to near normal levels and partial depletion of adherent cells provided varying degrees of relief of the observed suppression. Adherent cells (>95% macrophages) recovered from plastic surfaces were highly immunosuppressive. Suppressor cells appeared to interfere with both early and late stages of spleen cell proliferative responses. The results suggest that cells with some characteristics of macrophages within tissues near the maternal-fetal interface may create a local environment prohibitive to maternal lymphocyte proliferation.     

Accumulation and regulation of elastin in the rat uterus

The relative levels of elastin-specific mRNA were used as a measure of tropoelastin expression in uteri from pregnant Sprague-Dawley rats. The levels of elastin-specific mRNA were also correlated with values for net tropoelastin production and net deposition of mature, crosslinked elastin. The total content of uterine elastin increased throughout gestation, reaching maximal levels at Day 19 of gestation, which were three times those of nongravid tissue. Following involution, the elastin content decreased rapidly to near baseline values by 5 days postpartum. The content of soluble elastin, estimated using an enzyme-linked immunosorbent assay, paralleled in part the increase in elastin deposition and elastin mRNA levels. Uterine elastin metabolism appears to be unlike that in other elastic tissues, e.g., lung and large blood vessels. In most elastin containing tissues, the protein is synthesized during discrete developmental periods and is not readily degraded. However, uterine elastin is continuously expressed, and appears to be in a continual cycle of degradation and replacement.     

Characterization of murine erythropoietin receptor genes

We have isolated and characterized the murine genomic and complementary DNAs encoding erythropoietin (Epo) receptor from Epo-responsive and unresponsive mouse erythroleukemia cells. Two classes of Epo receptor cDNAs were isolated from Epo-responsive cells. One is a 55,000 Mr membrane-bound Epo receptor, and the other is a 29,000 Mr soluble Epo receptor lacking the transmembrane and cytoplasmic domains. As a result of alternative splicing, two insert sequences containing termination codons are produced, and the encoded polypeptide diverges four amino acids upstream from the transmembrane domain, adding 20 new amino acids before terminating. Amino acid sequence of the Epo receptor cDNA isolated from Epo-responsive cells was identical with that of Epo-unresponsive cells, indicating that Epo-responsiveness does not depend upon the primary structure of the Epo receptor (binding) protein. Analysis of 6.6 x 10(3) base-pairs (kb) genomic DNA segments covering complete Epo receptor gene and promoter regions revealed that potential regulatory elements (NF-E1, GF-1 or Eryf 1) for erythroid-specific and differentiation stage-specific gene expression are located in the promoter and 3' noncoding regions.