Fertility defects in Surfactant associated protein D knockout female mice: altered ovarian hormone profile

Surfactant associated protein D (SFTPD, also known as SP-D), a pattern recognition molecule, is an integral component of the mucosal immune system of female reproductive tract (FRT). In addition to host defense functions in the FRT, recent evidences indicate immunomodulatory role of SFTPD in parturition and pre-term labor. Regulation of SFTPD expression by ovarian hormones in the mouse uterus implicates SFTPD of FRT in pregnancy establishment and maintenance. In the current study, we attempted to decipher the functional relevance of SFTPD in FRT by characterizing the fertility parameters of surfactant associated protein D knockout (Sftpd^tm1Jhf/Sftpd^tm1Jhf) female mice. Knockout female mice exhibited extended estrous cycle with altered serum profile of ovarian hormones. We also demonstrate altered expression of ovarian hormone receptors and hormone responsive genes ITGB1, LIF and HOXA10 in uteri of these mice. Knockout females mated with wild type males had significantly smaller litter size due to increased pre-implantation embryo loss. We also observed an altered immune profile in knockout mice uteri with elevated levels of inflammatory cytokines, increased numbers of pro-inflammatory monocytes/macrophages and lower FOXP3 levels during the pre-implantation period. LPS administration to pregnant knockout mice did not result in any increase in embryo implantation loss and was associated with a blunted uterine pro-inflammatory response, plausibly due to higher levels of serum progesterone. Taken together, our results demonstrate that SFTPD deficiency affects female fertility, highlighting roles for SFTPD in ovarian and uterine physiology.     

The role of cytokines in the immune response to influenza A virus infection

Influenza A virus is one of the most important causes of respiratory tract diseases. It replicates in epithelial cells and leukocytes resulting in the production of immune mediators--cytokines, substances with various biological effects. Cytokines, as a part of innate immunity, favor the development of antiviral and TH 1-type immune responses. Cytokines also affect the adaptive immune response and disease manifestation. In the organism, the virus infection results in the production of chemotactic [a regulated upon activation, normal T cell-expressed and -secreted cytokine (RANTES), monocyte chemoattractant proteins (MCP) MCP-1, MCP-3, macrophage inflammatory protein 1 alpha (MIP- 1 alpha), interferon gamma-induced protein 10 (IP-10), and interleukin 8 (IL-8)], pro-inflammatory [IL- 1beta, IL-6, IL-18, and tumor necrosis factor alpha(TNF-alpha)] and antiviral [interferon (IFN) alpha/beta] cytokines. Whilst knowledge of the mechanisms underlying host and tissue specificity has advanced significantly, we still know relatively little about the function of cytokines released from different cells following influenza infection. In this review we deal with the role and mode of possible impact of cytokines on the disease pathogenesis and host immune response.     

Procoagulants in fetus rejection: the role of the OX-2 (CD200) tolerance signal.

The spontaneous loss of normal karyotype embryos may be initiated or prevented by the maternal immune system. In mice, loss between the time of implantation (day 4.5) and formation of a vascularized placenta (day 9.5) when the embryo is too large to survive by diffusion alone, is analogous to occult pregnancy failure in humans. They are called occult because usually the woman does not know she is pregnant. From studies in mice, these early losses have a different mechanism than abortion of a vascularized placenta (analogous to clinically evident human spontaneous miscarriage). The latter depend on the activation of the novel prothrombinase fgl2 on the fetal trophoblast and in maternal decidua by the T helper-1 (Th1) type cytokines TNF- alpha+gamma -interferon that arise from NK cells and NK gammadelta T cells; conversion of prothrombin to thrombin which in turn generates IL8 that activates polymorphonuclear leukocytes leads to embryonic death. These inflammatory processes are counteracted by Th2/3-type cytokines that arise in part from V gamma 1 delta 6 T cells reacting to, as yet, unidentified trophoblast antigens in the presence of the 'tolerance signaling molecule' OX-2. By contrast, peri-implantation losses (between implantation and formation of a vascularized placenta, analogous to occult losses in humans) appear to be dependent upon perforin(+)cells, complement activation, and products of alphabeta T and NK alphabeta T cells, but not on TNF- alpha or procoagulant activation. Similarities and differences between findings in the mouse and human, and the potential evolutionary significance of mechanisms affecting reproductive success are reviewed.     

Identification of possible mediators of embryonic mortality caused by mastitis: actions of lipopolysaccharide, prostaglandin F2alpha, and the nitric oxide generator, sodium nitroprusside dihydrate, on oocyte maturation and embryonic development in cattle

PROBLEM: Mastitis and immunization against constituents of organisms causing mastitis can reduce fertility of cattle and sheep, respectively. For the current experiments, it was hypothesized that these effects are mediated via actions of lipopolysaccharide (LPS), prostaglandin F2alpha (PGF2), and nitric oxide on oocyte maturation and embryonic development. METHOD OF STUDY: To evaluate effects on oocyte maturation, oocytes were matured with various concentrations of LPS, PGF2alpha, or the nitric oxide (NO) generator, sodium nitroprusside (SNP). Following maturation, oocytes were fertilized and cultured until day 8 after fertilization. To test effects on embryo growth, oocytes were matured and fertilized and cultured after fertilization with LPS, PGF2alpha, or SNP. RESULTS: Addition of 100 and 1000 ng/mL LPS and 50 and 100 ng/mL PGF2alpha to oocyte maturation medium reduced the proportion of oocytes that became blastocysts at day 8 after fertilization. When added after fertilization, in contrast, neither LPS nor PGF2alpha reduced development to the blastocyst stage. Unlike for LPS and PGF2alpha, addition of SNP during oocyte maturation was without effect on the proportion of oocytes that became blastocysts at day 8 after fertilization. However, addition of 10 microM SNP to culture medium after fertilization completely prevented development to the blastocyst stage while 0.1 and 1 microM SNP did not affect development. CONCLUSIONS: Results indicate that increased local concentrations of LPS, PGF2alpha, and NO can have deleterious consequences on oocyte function (LPS, PGF2alpha) and embryonic development (NO). Thus, these molecules are putative mediators of effects of infectious disease or inflammation, including mastitis, on fertility of cattle.     

Perlecan and syndecan-4 in uterine tissues during the early pregnancy in mice

PROBLEM: During early pregnancy in mice, there is recruitment of specific immune cells, remodeling of the endometrium, cell differentiation and synthesis of new molecules. METHOD OF STUDY: Immunohistochemistry was used to determine the distribution of perlecan and syndecan-4 in the uteri before and after embryo implantation. RESULTS: During pre-implantation, perlecan was identified in basement membranes and extracellular spaces of the endometrial stroma. In contrast, expression of syndecan-4 was quite weak. In the peri-implantation period, perlecan remained in the basement membranes, and it was no longer observed in the stroma and it was identified in the embryonic cells. On day 4 of pregnancy, syndecan-4 increased in the fibroblasts of the subepithelial stroma. After implantation, syndecan-4 was pronounced in pre-decidual and mature decidual cells. CONCLUSIONS: The coordinate balance between the pre- and post-implantation periods suggests a role of these two molecules in the adaptive modification of the uterine microenvironment to receive and implant the embryo.     

Effect of Escherichia coli and Lactobacillus rhamnosus on macrophage inflammatory protein 3 alpha, tumor necrosis factor alpha, and transforming growth factor beta release by polarized rat uterine epithelial cells in culture

Entry of bacteria from the vagina into the uterus raises the question of uterine epithelial cell (UEC) signaling in response to the presence of bacteria. Our model system helps to define microbially elicited UEC basolateral cytokine release, important in regulating underlying stromal immune cell protection. UECs from adult rats were grown in cell culture inserts to establish a confluent polarized monolayer as was determined by transepithelial resistance (TER). Polarized epithelial cell cultures were treated apically with live or heat-killed Escherichia coli or Lactobacillus rhamnosus prior to collection of basolateral media after 24 h of incubation. Coculture of polarized UECs with live E. coli had no effect on epithelial cell TER. In response to exposure to live E. coli, epithelial cell basolateral release of macrophage inflammatory protein 3 alpha (MIP3 alpha) and tumor necrosis factor alpha (TNF-alpha) increased at a time when basolateral release of biologically active transforming growth factor beta (TGF-beta) decreased. Incubation of UECs with heat-killed E. coli resulted in an increased basolateral release of MIP3 alpha and TNF-alpha, without affecting TER or TGF-beta. In contrast to E. coli, live or heat-killed L. rhamnosus had no effect on TER or cytokine release. These studies indicate that polarized rat UECs respond to gram-negative E. coli by releasing the cytokines MIP3 alpha and TNF-alpha, signals important to both the innate and adaptive immune systems. These findings suggest that UEC responses to bacteria are selective and important in initiating and regulating immune protection in the female reproductive tract.     

Evidence that obesity alters the quality of oocytes and embryos

Infertility is more common in overweight and obese women, with reproductive impairments occurring at many levels of the hypothalamic-ovarian-uterine axis. These impairments lead primarily to longer times to conception and decreased pregnancy rates and have resulted in increasing numbers of overweight and obese women seeking assisted reproduction technologies, such as in vitro fertilization or IVF. Even after undertaking IVF procedures obese women have decreased pregnancy rates compared to moderate weight women, suggesting there may be intrinsic differences in the oocytes of these patients. Definitive data is lacking however, and thus the effect of obesity on oocyte quality remains one of the biggest controversies in reproductive medicine. This review summarizes the studies to date which have yielded information about the effects of obesity on human oocyte quality and pre-implantation embryo development. In addition recent results from our laboratory which clearly demonstrate that diet-induced obesity in mice impairs oocyte developmental competence are discussed.     

Immunomodulatory function of seminal catecholamines may be an adaptation for reproduction

Catecholamines are found at high concentrations in seminal fluid. The exact functional significance of seminal catecholamines is unknown. We hypothesize that seminal catecholamines perform important immunomodulatory functions that support reproductive success. Specifically, we propose that catecholamines contribute to a local adaptive shift of T helper (Th) balance to Th2 dominance in the maternal reproductive tract to enable the gametes, and possibly the nascent zygote, to evade immune surveillance of the female. Our hypothesis suggests that the Th2 effects of catecholamines are independent of the direct immunomodulatory effects of seminal cytokines such as prostaglandin E2 and transforming growth factor beta1. Potential immunomodulatory functions of other seminal constituents such as aldosterone, oxytocin, vasopressin, and angiotensin remain unexplored and represents a topic of future interest. Seminal stress hormones may play a role in mating dynamics since alpha males typically live in a state of high hormonal stress, Mating with adrenalized alpha males may represent an adaptive Darwinian strategy by females to maximize their reproductive fitness.     

Cytokine and Toll-like receptor mRNAs in the nasal-associated lymphoid tissues of cattle during foot-and-mouth disease virus infection

The pharyngeal region is known to play an important role in foot-and-mouth disease virus (FMDV) infection in relation to acute disease and viral persistence. In this study, the local mucosal immune response in nasal-associated lymphoid tissue (NALT) of cattle infected with FMDV (strain O UKG 34/2001) was examined. Quantitative "real-time" RT-PCR assays were used to measure mRNA expression of cytokines (IFN-alpha, beta and gamma, IL-2, IL-1alpha and TNF-alpha) and Toll-like receptors (TLR)-3 and -4. NALTs from dorsal soft palate were collected from cattle at 7 days post-infection (dpi) and from carriers and non-carriers at 64 dpi. Expression of IFN-alpha mRNA was significantly greater in NALT during acute disease than in uninfected animals. Increased expression of IFN-gamma and IL-1alpha mRNA was also observed but was much lower than IFN-alpha expression. There was a slight increase in mRNA expression of TNF-alpha and IL-2. During persistence, TNF-alpha mRNA expression in carrier cattle was much higher than in non-carrier cattle. Expression of TLR-4 in NALT during the acute stage of infection was greater than in uninfected animals. Carrier and non-carrier cattle did not differ in respect of expression of TLR-3 and -4 mRNA in NALT.     

Endometrial markers of uterine receptivity utilizing the donor oocyte model

BACKGROUND: Ethical constraints limit the ability to study peri-implantation phase human endometrium. In this study, the donor oocyte model was used to study candidate endometrial markers of uterine receptivity. METHODS: Archived, paraffin-embedded tissue obtained by endometrial biopsy during cycle days 21-23 of patients undergoing 'mock' hormonal treatment cycles were evaluated by standard histological criteria and immunohistochemical staining for alpha v beta 3 integrin and glycodelin. All of these patients (n = 101) had undergone a donor oocyte embryo transfer cycle utilizing the exact same hormonal protocol. RESULTS: Histological evaluation revealed 62 (61.3%) in-phase, 34 (33.7%) dyssynchronous, 2 (2.0%) immature and 3 (3.0%) advanced endometria. The clinical outcomes of patients with either in-phase or dyssynchronous endometria were similar. Very strong correlations were noted between endometrial glandular dating and either alpha v beta 3 integrin or glycodelin immunostaining intensity (P < 0.001 for both). Glycodelin and alpha v beta 3 integrin immunostaining intensities were also highly correlated with each other (P < 0.001). CONCLUSIONS: Throughout the time period corresponding to the putative window of maximal endometrial receptivity (cycle days 21-23) a dynamic process was observed in exogenous hormonal replacement cycles characterized by a rapid histological advancement of endometrial glandular elements as well as progressive alpha v beta 3 integrin and glycodelin expression.     

Secretion of cytokines and chemokines by polarized human epithelial cells from the female reproductive tract

BACKGROUND: Pro-inflammatory chemokines that attract and cytokines that activate immune cells contribute to normal physiological homeostasis in the female reproductive tract, and are needed to deal effectively with potential pathogenic microbes. Mucosal epithelial cells are capable of producing these factors that communicate with cells of the innate and adaptive immune systems. METHODS: Epithelial cells from Fallopian tube, endometrium and endocervix were isolated and grown to high transepithelial resistance in cell inserts from seven patients who had hysterectomies. Interleukin (IL)-8, IL-6, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) and macrophage inflammatory peptide-1beta (MIP-1beta) were assessed by Luminex bead analysis or enzyme-linked immunosorbent assay (ELISA) in epithelial cell conditioned media from the apical and basolateral compartments. RESULTS: With the exception of MCP-1, the seven chemokines/cytokines constitutively produced by the polarized epithelial cells were preferentially secreted apically. A concentration pattern was found in all cases, with IL-8 and IL-6 produced in the greatest quantity. CONCLUSIONS: The concentrations of IL-8, IL-6, G-CSF and MCP-1 are similar to the levels found in reproductive tract fluids of patients with infection. The constitutive secretion and compartmentalization of large quantities of bioactive chemokines and cytokines provide additional evidence for the role of epithelial cells as gatekeepers of innate immune protection in the female reproductive tract.     

Lipopolysaccharide from an Escherichia coli htrB msbB mutant induces high levels of MIP-1 alpha and MIP-1 beta secretion without inducing TNF-alpha and IL-1 beta

OBJECTIVE: To identify a lipopolysaccharide (LPS) that retains the capacity to induce beta-chemokine secretion without the concomitant activation of pyrogenic cytokines. METHODS: LPS was extracted from strain MLK986 (mLPS), an htrB1::Tn10, msbB::ocam mutant of Escherichia coli that is defective for lipid A synthesis, and from wild-type parent E coli strains, W3110 (wtLPS). The capacity of these LPS preparations to induce tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory proteins 1 alpha (MIP-1 alpha) and MIP-1 beta was assessed using a human peripheral blood mononuclear cell (PBMC) activation assay. RESULTS: Stimulation of PBMCs with mLPS did not induce measurable levels of pyrogenic cytokines TNF-alpha and IL-1 beta, whereas wtLPS induced high levels of these cytokines. Furthermore, mLPS antagonized the induction of TNF-alpha secretion by wtLPS. Nonetheless, mLPS retained a discrete agonist activity that induced MIP-1 alpha and MIP-1 beta secretion by PBMCs. This latter agonist activity appears to be unique to mLPS, since two previously documented LPS antagonists, Rhodobacter sphaeroides diphosphoryl lipid A and synthetic lipid IVA, did not induce MIP-1 alpha and MIP-1 beta secretion. Furthermore, synthetic lipid IVA was an antagonist of MIP-1 alpha and MIP-1 beta induction by mLPS. CONCLUSION: These results show that mLPS exhibits a novel bipartite activity, being an effective antagonist of TNF-alpha induction by wtLPS, while paradoxically being an agonist of MIP-1 alpha and MIP-1 beta secretion.     

Primary role of interleukin-1 alpha and interleukin-1 beta in lipopolysaccharide-induced hypoglycemia in mice

Within a few hours of its injection into mice, lipopolysaccharide (LPS) induces hypoglycemia and the production of various cytokines. We previously found that interleukin-1 alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) induce hypoglycemia and that the minimum effective dose of IL-1 alpha or IL-1 beta is about 1/1000 that of TNF-alpha. In the present study, we examined the contribution made by IL-1 to the hypoglycemic action of LPS. Nine other cytokines tested were all inactive at inducing hypoglycemia. LPS produced hypoglycemia in mice deficient in either IL-1 alpha or IL-1 beta but not in mice deficient in both cytokines (IL-1 alpha and -1 beta knockout [IL-1 alpha/beta KO] mice). IL-1 alpha, IL-1 beta, and TNF-alpha induced hypoglycemia in IL-1 alpha/beta KO mice, as they did in normal control mice. The LPS-induced elevation of serum cortisol was weaker in IL-1 alpha/beta KO mice than in control mice, and, in the latter, serum cortisol was markedly raised while blood glucose was declining. IL-1 alpha decreased blood glucose both in NOD mice (which have impaired insulin production) and in KK-Ay mice (insulin resistant). These results suggest that (i). cortisol may not be involved in mediating the resistance of IL-1 alpha/beta KO mice to the hypoglycemic action of LPS, (ii). as a mediator, IL-1 is a prerequisite for the hypoglycemic action of LPS, (iii). IL-1 alpha and IL-1 beta perform mutual compensation, and (iv). IL-1 plays a role as the primary stimulator of the many anabolic reactions required for the elaboration of immune responses against infection.     

Cultured human monocytes secrete fibronectin in response to activation by proinflammatory cytokines

We studied the effects of the cytokines IL-1alpha, IL-6, tumour necrosis factor-alpha (TNF-alpha), IL-4, IL-10, IL-13 and transforming growth factor-beta (TGF-beta) on fibronectin (FN) production by cultured-human monocytes. IL-1alpha, IL-6 and TNF-alpha all increased FN production, an indicator of monocyte activation. These cytokines increased FN production in a dose-dependent fashion, with a 4-h treatment being sufficient to measure FN production by radioimmunoassay. Conversely, IL-4, IL-10 and IL-13 strongly inhibited cytokine-induced FN production, while TGF-beta only partially inhibited FN production. The combination of suboptimal doses of cytokines (IL-1alpha + IL-6, IL-1alpha + TNF-alpha, IL-6 + TNF-alpha), which could not singly induce substantial amounts of FN, were able to induce FN production by cultured monocytes. Northern blot analysis with a cDNA specific for FN confirmed the expression of FN mRNA in cultured monocytes stimulated with a single cytokine or a combination of cytokines. Our data demonstrate that monocytes may not always require high concentrations of cytokines for activation in vitro, and that the synergistic or additive action of low levels of cytokines on monocyte activation may be sufficient to promote immune or inflammatory reactions. Our data also suggest that certain T cell cytokines may regulate monocyte activation.     

Dysregulation of the Cytokine Network in the Uterus of the Diabetic Rat

Insulin-dependent (type I) diabetes is an auto-immune disorder that produces secondary complications in numerous non-immunological systems. Changes in the synthesis and action pattern of several cytokines have been associated with the development of these alterations. Based on the clinical facts that the pregnant and non-pregnant functions of the reproductive system are also disrupted by diabetes, our laboratory has decided to concentrate its research activities on the hypothesis that cytokines may be implicated in the uteropathy and embryopathy associated with the metabolic disorder. This review article summarizes our major findings concerning the synthesis of TNF-alpha and IL-1beta in the uterus of diabetic rats, and in cultures of rodent uterine cells upon their exposure to high concentrations of glucose. The paper also reviews evidence that both the peri-implanting embryo and the epithelial cell layer lining the uterine lumen are targets for the deleterious influence of excess TNF-alpha. If confirmed in the uterus of diabetic patients, these observations may explain how cytokines contribute to the dysregulation of crucial reproductive events like menstruation and embryo implantation in humans.     

Dissociation between plasma and monocyte-associated cytokines during sepsis

We report our investigations of circulating interleukin (IL) 1 beta, IL 6 and tumor necrosis factor (TNF)-alpha, as well as cell-associated IL 1 alpha, IL 1 beta and TNF-alpha in plasma and monocytes of 21 patients with sepsis syndrome and 6 patients with non-septic shock. Longitudinal studies reveal that (a) the most frequent detectable plasma cytokines were TNF-alpha and IL 6, (b) the presence and the kinetics of circulating cytokines were independent of one other, (c) detectable levels of cytokines could be found for a long period of time, and (d) significantly higher levels of IL 6 were found for non-surviving patients. Because of the in vivo half-life of cytokines and of the existence of numerous specific high-affinity receptors, it is quite probable that detectable plasma cytokines represent the excess of produced mediators which have not been trapped by the target cells. TNF-alpha (410 +/- 65 pg/10(6) monocytes) and IL 1 beta (153 +/- 60 pg/10(6) monocytes) were frequently found associated to monocyte lysates (88% and 50%, respectively). Despite the fact that IL 1 alpha is the most abundant cytokine found associated to monocytes following in vitro activation, IL 1 alpha was rarely found in monocytes of intensive care unit patients (29%). No correlation was found to exist between the levels of plasma cytokines and cell-associated cytokines. Some patients had plasma TNF-alpha or IL 1 beta in the absence of the corresponding monocyte-associated cytokine. This observation suggests that cells other than monocytes can participate in the production of circulating cytokines. At the end of the longitudinal study (day 14 +/- 2), only 2/12 surviving patients still had plasma TNF-alpha, whereas 8/12 had monocyte-associated TNF-alpha. These results indicate that activation of monocytes still occurs in patients for whom no plasma cytokines can be detected. Thus, in addition to the measurement of plasma cytokine, measurement of cell-associated cytokine appears useful to assess cytokine production and monocyte activation in vivo.