Concomitant elevations of MMP‐9, NGAL,proMMP‐9/NGAL and neutrophil elastase in serum of smokers with chronic obstructive pulmonary disease

A growing body of evidence points towards smoking‐related phenotypic differences in chronic obstructive pulmonary disease (COPD). As COPD is associated with systemic inflammation, we determined whether smoking status is related to serum levels of matrix metalloproteinase‐9 (pro‐ and active MMP‐9), neutrophil gelatinase‐associated lipocalin (NGAL) and the proMMP‐9/NGAL complex in patients with COPD. Serum samples were collected in 100 stable‐phase COPD patients (82 smokers, 18 never‐smokers) and 28 healthy adults (21 smokers, 7 never‐smokers). Serum levels of studied factors were measured in ELISA. Our data provide the first evidence of simultaneously elevated serum levels of MMP‐9, NGAL and proMMP‐9/NGAL in COPD smokers. While the triad discriminated between smokers and non‐smokers in the COPD group, MMP‐9 and proMMP‐9/NGAL (but not NGAL) discriminated between smokers with and without COPD. Adjustment for age and smoking pack‐years did not alter the findings. Serum MMP‐9, NGAL and proMMP‐9/NGAL levels were not correlated with the GOLD stage or FEV1 decline. Furthermore, serum levels of neutrophil elastase (NE) and MMP‐3 (but not of IL‐6 and MMP‐12) were also higher in COPD smokers than in healthy smokers before and after adjustment for age and pack‐years. Among COPD smokers, levels of MMP‐9, NGAL and proMMP‐9/NGAL were positively correlated with NE (P < 0.0001) but not with the remaining factors. Gelatin zymography detected proMMP‐9 in serum samples of healthy and COPD smoking groups. Our results suggest that associated serum levels of proMMP‐9, NGAL, proMMP‐9/NGAL and NE may reflect the state of systemic inflammation in COPD related to cigarette smoking.     

Activation of macrophage promatrix metalloproteinase-9 by lipopolysaccharide-associated proteinases

LPS induces an up-regulation of promatrix metalloproteinase-9 (proMMP9) gene expression in cells of the monocyte/macrophage lineage. We demonstrate here that LPS preparations are also able to activate proMMP9 made by human macrophages or THP-1 cells via LPS-associated proteinases, which cleave the N-terminal propeptide at a site or sites close to the one cleaved upon activation with organomercurial compounds. LPS-associated proteinases are serine proteinases that are able to cleave denatured collagens (gelatin) and the mammalian serine proteinase inhibitor, alpha(1)-proteinase inhibitor, thereby pushing the balance of extracellular matrix turnover even further toward degradation. A low molecular mass, low affinity inhibitor of MMP9, possibly derived from the propeptide, is generated during proMMP9 activation. However, inhibition of the LPS-associated proteinases had no effect on proMMP9 synthesis, indicating that their proteolytic activity was not required for signaling the up-regulation of the proMMP9 gene.     

Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation

Recent epidemiological studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, matrix metalloproteinase 9 (MMP9) is implicated in the invasion and metastasis of tumour cells. We examined the involvement of Porphyromonas gingivalis, a periodontal pathogen, in OSCC invasion through induced expression of proMMP and its activation. proMMP9 was continuously secreted from carcinoma SAS cells, while P. gingivalis infection increased proenzyme expression and subsequently processed it to active MMP9 in culture supernatant, which enhanced cellular invasion. In contrast, Fusobacterium nucleatum, another periodontal organism, failed to demonstrate such activities. The effects of P. gingivalis were observed with highly invasive cells, but not with the low invasivetype. P. gingivalis also stimulated proteinase‐activated receptor 2 (PAR2) and enhanced proMMP9 expression, which promoted cellular invasion. P. gingivalis mutants deficient in gingipain proteases failed to activate MMP9. Infected SAS cells exhibited activation of ERK1/2, p38, and NF‐kB, and their inhibitors diminished both proMMP9‐overexpression and cellular invasion. Together, our results show that P. gingivalis activates the ERK1/2‐Ets1, p38/HSP27, and PAR2/NF‐kB pathways to induce proMMP9 expression, after which the proenzyme is activated by gingipains to promote cellular invasion of OSCC cell lines. These findings suggest a novel mechanism of progression and metastasis of OSCC associated with periodontitis.     

Seminal plasma regulates corpora lutea macrophage populations during early pregnancy in mice

In mice, exposure of the uterus to seminal plasma at mating initiates an inflammatory response within the endometrium, which is characterized by production of cytokines that recruit and activate leukocytes. We hypothesized that this seminal plasma-induced inflammatory response would extend to the ovary, increasing leukocyte abundance within corpora lutea and potentially enhancing progesterone synthesis. Female mice mated to males with their seminal vesicles surgically removed exhibited fewer macrophages within corpora lutea on the day after mating, compared with females mated to vasectomized or normal, intact males. The mean number of F4/80-positive macrophages and major histocompatibility complex (MHC) class II-positive activated macrophages was approximately 2-fold fewer in the absence of seminal vesicle fluid. The effects of seminal plasma on macrophage abundance subsided by Day 4 and were not accompanied by a change in serum progesterone levels during luteinization (Days 1, 2, or 4 after mating) or luteolysis (Days 6 or 9). In vitro secretion of progesterone from corpora lutea cultured with or without LH also did not differ between treatment groups. There was no effect of seminal plasma deficiency in males on the number of ovulated ova or corpora lutea in females. These results imply that seminal plasma exposure of the female reproductive tract at mating augments the macrophage population of newly formed corpora lutea, although these additional macrophages seem not to play a role in steroidogenesis and may instead be involved in tissue remodeling within corpora lutea.     

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice,and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross‐talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G‐protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G‐protein activation, plays an important role in embryo implantation by interfering in the cross‐talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17β‐estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17β‐estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17β‐estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.     

Membrane type 1 matrix metalloproteinase and gelatinase A synergistically degrade type 1 collagen in a cell model

A fibrosarcoma cell line transfected with the matrix metalloproteinase MT1 MMP showed an enhanced ability to degrade 14C-labelled collagen films. As previously shown for proMMP 2 activation, TIMP 1 was an ineffective inhibitor of the process of collagenolysis whereas TIMP 2 was efficient and completely prevented collagen degradation. In the presence of the calcium ionophore, ionomycin, proteolytic processing of MT1 MMP was restricted and collagenolysis did not occur indicating that the 63 kDa form of the enzyme is not a functional collagenase. The collagenolytic activity of MT1 MMP was shown to be enhanced by the addition of proMMP 2, but TIMP 1 inhibition remained poor relative to that of TIMP 2. The study demonstrated that synergy between two non-conventional collagenases effectively degrades insoluble pericellular collagen. Due to the membrane localisation of MT1 MMP, this could potentially occur in a highly localised manner.     

Distribution of Bandeiraea simplicifolia lectin binding sites in the genital organs of female and male mice

Fluorescein isothiocyanate labelled type I lectin from Bandeiraea simplicifolia (BSA-I) known for its specific binding to alpha-D-galactopyranosyl and 2-acetamido-2-deoxy-D-galactose groups, has been used to map the distribution of the lectin specific binding sites in the genital organs of female and male mice. In non-pregnant female mice, strong lectin reactivity was restricted to the epithelium of the distal oviduct, the cervix and vagina. In pregnant mice strong BSA-I reactivity was also noted in the epithelium of uterine glands from the time of implantation on day 5 onward. In the testis BSA-I bound selectively to sperm but did not react with other cells in the seminiferous tubules. In the proximal caput epididymis BSA-I reacted with the epithelial cells, the underlying basement membranes and the intraluminal sperm. The intraluminal contents of the seminal vesicles reacted strongly with the lectin. Our data thus show a widespread but selective distribution of BSA-I lectin binding sites in the male and female genital organs and altered lectin binding in the uterus during pregnancy.     

Progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus

Populations of macrophages and neutrophils in the uterus are under the control of the female sex steroids estrogen and progesterone (P4). Their influx is induced by estrogen, while P4 can both stimulate and inhibit leukocyte influx depending on the timing of P4 with respect to estrogen. Regulation of leukocytes has been implicated in changes in uterine immune responses during the estrous cycle, pregnancy, and implantation. This work demonstrates that P4 given concurrently with estrogen to ovariectomized mice for 4 days antagonizes the ability of estrogen to recruit macrophages and neutrophils into the mouse uterus. Using progesterone receptor knockout (PRKO) mice, we show that this effect is dependent on progesterone receptors (PR). In the absence of PR, neutrophils recruited by estrogen were found to be degranulated, partially explaining the edema that is observed with long-term treatment of PRKO mice with estrogen and P4. Populations of B lymphocyte cells were shown to be unchanged by estrogen and P4 treatment in both wild-type and PRKO mice. The neutrophil chemotactic chemokine MIP-2 was examined for down-regulation by P4 but was found to be unaffected by hormonal treatment. Together, these observations demonstrate that PR has a strong anti-inflammatory role in the mouse uterus when estrogen and P4 are present together.     

Matrix metalloproteinases in endometrial breakdown and repair: functional significance in a mouse model

Considerable correlative evidence suggests an important role for matrix metalloproteinases (MMPs) in menstruation, a process which occurs naturally in very few species. In this study, MMP expression was examined in a mouse model of endometrial breakdown and repair and the functional importance of MMPs determined. In the model, progesterone support was withdrawn from mice in which endometrial decidualization had been induced; 24 h later, endometrial breakdown was complete, and the entire decidual zone had been shed. Re-epithelialization had occurred by 36 h, and the endometrium had undergone extensive restoration toward a predecidualized state by 48 h. Immunoreactive MMP9 and MMP7 colocalized with leukocyte subsets, particularly neutrophils, whereas MMP13 staining was always extracellular. MMP3 and MMP7 were abundant during re-epithelialization in close proximity to newly reforming epithelium. The functional importance of MMPs in these processes was examined using two MMP inhibitors, doxycycline and batimistat. Both inhibitors effectively reduced MMP activity, as assessed by in situ zymography, but did not have significant effects on endometrial breakdown or repair. This study demonstrates that although MMPs are present in abundance during endometrial breakdown and repair in this mouse model, they are not the key mediators of these processes.     

Molecular mechanisms of the antihormonal and antiimplantation effects of norethisterone and its a-ring reduced metabolites

Norethisterone (NET) has been used as a contragestational postcoital agent. It is biotrans-formed to 5α dihydro-NET (5α-NET) and 3β,5α tetrahydro-NET (3β,5α-NET) in target tissues. The participation of these metabolites in NET effects is unknown. We have examined the antiimplantation and antiprogestational effects of NET and its metabolites, in adult mated female rabbits, by assessing the number of implantation sites and the expression products of the uteroglobin (UTG) gene in the uterus, and by comparing them with those of RU-486 and estradiol. Steroids were daily administered s.c. at several doses for 7 consecutive days, starting 24 hr after coitus. To assure that fertilization occurred in all animals, the presence of early pregnancy factor was determined. The results demonstrated that high doses (5 mg/kg) of NET reduced both implantation and the expression of the UTG gene. On the other hand, lower doses (1.5 mg/kg) of 5α-NET produced an antiimplantation effect and suppressed UTG synthesis and its mRNA. These effects were similar to those of RU-486. At lower doses (1 mg/kg), both estradiol and the estrogenic metabolite 3β,5α-NET were also effective in inhibiting implantation and UTG gene expression. The overall results suggest that NET metabolites exert antiimplantation and antiprogestational effects through their interaction with progesterone and estrogen receptors, and provide an explanation for the molecular mechanisms involved in the postcoital contraceptive action of NET. © 1995 Wiley-Liss, Inc.     

Sex hormone changes induced by the parasite lead to feminization of the male host in murine Taenia crassiceps cysticercosis

Female mice are more susceptible to Taenia crassiceps (TC) infection than males. However, after a month parasite load increases massively in both genders reaching thousands of parasites per host. The possibility of hormonal changes in the infected mice was envisaged. Sex hormones levels were assayed after different periods of infection, the parasites present in the peritoneal cavity were collected and gonads, uterus and seminal vesicles were weighed. In male mice, serum estradiol increased to levels 200 times their normal values whilst those of testosterone decreased 90% relative to controls. The weight of seminal vesicles was significantly diminished. Infected female mice also showed a slight increase in estrogen blood levels after 8 weeks of infection and the weight of the uterus was significantly increased relative to controls. Serum estradiol and testosterone were almost undetectable after gonadectomy. Cytokines such as IL-6 are capable of stimulating aromatase activity and we found that splenocytes from infected mice produced amounts of IL-6 higher than control as measured by ELISA. In conclusion T. crassiceps infection triggers a feminization process in the infected hosts. The gonads are required for the parasite to induce higher estrogen synthesis. IL-6 could be involved in the immunoendocrine mechanism used by the parasite to maintain a highly permissive environment for its rapid growth.     

Matrix Metalloprotease 9 Mediates Neutrophil Migration into the Airways in Response to Influenza Virus-Induced Toll-Like Receptor Signaling

The early inflammatory response to influenza virus infection contributes to severe lung disease and continues to pose a serious threat to human health. The mechanisms by which neutrophils gain entry to the respiratory tract and their role during pathogenesis remain unclear. Here, we report that neutrophils significantly contributed to morbidity in a pathological mouse model of influenza virus infection. Using extensive immunohistochemistry, bone marrow transfers, and depletion studies, we identified neutrophils as the predominant pulmonary cellular source of the gelatinase matrix metalloprotease (MMP) 9, which is capable of digesting the extracellular matrix. Furthermore, infection of MMP9-deficient mice showed that MMP9 was functionally required for neutrophil migration and control of viral replication in the respiratory tract. Although MMP9 release was toll-like receptor (TLR) signaling-dependent, MyD88-mediated signals in non-hematopoietic cells, rather than neutrophil TLRs themselves, were important for neutrophil migration. These results were extended using multiplex analyses of inflammatory mediators to show that neutrophil chemotactic factor, CCL3, and TNFα were reduced in the Myd88 ^−/− airways. Furthermore, TNFα induced MMP9 secretion by neutrophils and blocking TNFα in vivo reduced neutrophil recruitment after infection. Innate recognition of influenza virus therefore provides the mechanisms to induce recruitment of neutrophils through chemokines and to enable their motility within the tissue via MMP9-mediated cleavage of the basement membrane. Our results demonstrate a previously unknown contribution of MMP9 to influenza virus pathogenesis by mediating excessive neutrophil migration into the respiratory tract in response to viral replication that could be exploited for therapeutic purposes.     

Lymphoid tissue changes during early syngeneic pregnancy in the RT1u rat.

An active immunological response by the female to mating and pregnancy is important, if not essential, to successful reproduction. Alloantigens which have been demonstrated on spermatozoa include histocompatibility antigens. In the enigmatic success of the fetal allograft, the conceptus survives owing to failure of the mother either to initiate or complete the process of rejection. In this study, AO(RT1u/AgB2) female rats were caged with male rats of the same strain. Observations were obtained from the first 5 days of their postcoital period, identifying lymphoid tissue changes which are due to the presence of the ejaculate and early products of conception in the female reproductive tract. A significant increase in intrathymic proliferation by day 3 is thought to be a hormonal effect induced by the cervical stimulation at mating. The postcoital exodus of lymphocytes from the thymus is possibly in response to coitus and induced by the seminal plasma to safeguard the implanting conceptus.     

TGF superfamily and MMP2, MMP9, TIMP1 genes expression in the endometrium of women with impaired reproduction

During the putative "implantation window", a period of maximal endometrial receptivity that spans 7-9 days after ovulation, a series of changes on the structural and molecular level occur that render the endometrium susceptible to implantation for the human embryo. Many members of the TGFbetas are expressed by human endometrium at different stages of menstrual cycle. Also studies regarding the MMP2 gene expression and activity of MMP2 in the implantation window have shown a higher expression and activity of MMP2 in women with impaired fertility. We have examined by RT-PCR the expression of TGFbeta2 and MMP2, MMP9 and TIMP1 in 28 patients with idiopathic infertility, 16 patients with unexplained recurrent miscarriage and 16 control women were enrolled in this study. Seven to nine days after ovulation endometrial biopsy by Pipelle or hysteroscopy was performed to assess the expression of TGFbeta2 , MMP2, MMP9 and TIMP1. We found that in endometria from women with idiopathic infertility TGFbeta2 expression was 2.8 fold higher than in endometria from control group and 2.1 fold higher in endometrial samples from women with unexplained recurrent miscarriage compared to the control group. The MMP2, MMP9 and TIMP1 expression in endometrial samples revealed no significant differences between the study groups and control group. There was a statistically significant negative correlation between TGFbeta2 and MMP9 expression in endometria from women in control group. The present investigations suggest that dysregulated TGFbeta2, MMP2, MMP9 and TIMP1 expression are associated with infertility and early pregnancy loss. However the exact mechanism of how overexpression of endometrial TGFbetaand MMPs interferes with implantation may be more complex.     

Distribution of Bandeiraea simplicifolia lectin binding sites in the genital organs of female and male mice

Summary Fluorescein isothiocyanate labelled type I lectin from Bandeiraea simplicifolia (BSA-I) known for its specific binding to -d-galactopyranosyl and 2-acetamido-2-deoxy-d-galactose groups, has been used to map the distribution of the lectin specific binding sites in the genital organs of female and male mice. In non-pregnant female mice, strong lectin reactivity was restricted to the epithelium of the distal oviduct, the cervix and vagina. In pregnant mice strong BSA-I reactivity was also noted in the epithelium of uterine glands from the time of implantation on day 5 onward. In the testis BSA-I bound selectively to sperm but did not react with other cells in the seminiferous tubules. In the proximal caput epididymis BSA-I reacted with the epithelial cells, the underlying basement membranes and the intraluminal sperm. The intraluminal contents of the seminal vesicles reacted strongly with the lectin. Our data thus show a widespread but selective distribution of BSA-I lectin binding sites in the male and female genital organs and altered lectin binding in the uterus during pregnancy.     

Overexpression of MMP9 in macrophages attenuates pulmonary fibrosis induced by bleomycin

Pulmonary fibrosis is a common response to a variety of lung injuries, characterized by fibroblast/myofibroblast expansion and abnormal accumulation of extracellular matrix. An increased expression of matrix metalloprotease 9 (MMP9) in human and experimental lung fibrosis has been documented, but its role in the fibrotic response is unclear. We studied the effect of MMP9 overexpression in bleomycin-driven lung fibrosis using transgenic mice expressing human MMP9 in alveolar macrophages (hMMP9-TG). At 8 weeks post-bleomycin, the extent of fibrotic lesions and OH-proline content were significantly decreased in the TG mice compared to the WT mice. The decreased fibrosis in hMMP9-TG mice was preceded by a significant reduction of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) at 1 and 4 weeks post-bleomycin, respectively, as well as by significantly less TIMP-1 than the WT mice. From a variety of cytokines/chemokines investigated, we found that BAL levels of insulin-like growth factor binding protein-3 (IGFBP3) as well as the immunoreactive protein in the lungs were significantly lower in hMMP9-TG mice compared with WT mice despite similar levels of gene expression. Using IGFBP-3 substrate zymography we found that BAL from TG mice at 1 week after bleomycin cleaved IGFBP-3. Further, we demonstrated that MMP9 degraded IGFBP-3 into lower molecular mass fragments. These findings suggest that increased activity of MMP9 secreted by alveolar macrophages in the lung microenvironment may have an antifibrotic effect and provide a potential mechanism involving IGFBP3 degradation.     

Seminal plasma and male factor signalling in the female reproductive tract

In mammals, insemination results in the transmission of seminal factors that act, in the female reproductive tract, to promote sperm survival, to “condition” the female immune response to tolerate the conceptus and to organise molecular and cellular changes in the endometrium to facilitate embryo development and implantation. These events are initiated when signalling agents, including transforming growth factor-β and other cytokines and prostaglandins secreted by seminal vesicle and prostate glands, interact with epithelial cells in the cervix and uterus to activate cytokine synthesis and to induce cellular and molecular changes resembling a classical inflammatory cascade. The consequences are the recruitment and activation of macrophages, granulocytes and dendritic cells, which have immune-regulatory and tissue-remodelling roles that culminate in improved endometrial receptivity to the implanting embryo. Cytokines elicited by seminal activation have embryotrophic properties and also contribute directly to the optimal development of the early embryo. This review summarises our current understanding of the physiology of responses to seminal plasma in the female reproductive tract and considers the evolutionary significance of seminal plasma in influencing female tissues to promote the success of pregnancy.The author acknowledges the support of the NHMRC of Australia Fellowship and Program Grant schemes.     

Matrix metalloproteinase expression correlates with virulence following neurotropic mouse hepatitis virus infection

The relationship(s) between viral virulence and matrix metalloproteinase (MMP) expression in the central nervous system (CNS) of mice undergoing lethal and sublethal infections with neurotropic mouse hepatitis virus was investigated. Lethal infection induced increased levels of MMP-3 and MMP-12 mRNAs as well as that of tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) compared to sublethal infection. Increased induction of MMP, TIMP, and chemokine expression correlated with increased virus replication but not with inflammatory cell infiltration. Infection of immunosuppressed mice suggested that expression of most MMP, TIMP, and chemokine mRNA was induced primarily in CNS-resident cells. By contrast, MMP-9 protein activity was associated with the infiltration of neutrophils into the CNS. These data indicate an association between the magnitude of inflammatory gene expression within the CNS and viral virulence.     

Long-term exposure of male and female mice to 50 Hz magnetic field: effects on fertility

The effect of an extremely low frequency (ELF) magnetic field on the fertility of adult male and female Swiss mice was investigated. Adult male and female mice were exposed to a 50 Hz sinusoidal magnetic field at approximately 25 microT (rms) for 90 days before they were mated with unexposed counterparts. There were no exposure related effects on the fertility of male or female mice. The number of implantation sites, viable fetuses, and the total number of resorptions were not significantly affected in females impregnated by males exposed to the 50 Hz magnetic field as compared with the control group. The number of implantation sites, viable fetuses and the total number of resorptions in exposed females were also not statistically different from the control group. There were no significant effects on the weights of the testes, seminal vesicles, preputial gland or body weights of males exposed to 50 Hz magnetic field. Furthermore, body and uterine weights were not affected in females exposed to 50 Hz field; however, ovarian weight was significantly increased in females exposed to the same field. These results suggest that exposure of male and female mice to low frequency magnetic field had no adverse effects on fertility and reproduction in mice.