Characterization of the innate immune response in goats after intrauterine infusion of E. coli using histopathological, cytologic and molecular analyses

The objective was to characterize the innate immune response in dairy goats after intrauterine infusion of E. coli. A suspension of Escherichia coli (E. coli; 4 × 10⁹ cfu (cfu)/mL; experimental group, n = 6) or 5 mL PBS (control group, n = 6) were infused once into each uterine horn in goats at 25 days postpartum. Blood and endometrial biopsy samples were collected preinoculation (0 h) and at 3, 6, 12, 24, 72, 120, and 168 h post inoculation (pi). Relative gene expression analyses of Toll-like receptor4 (TLR4), tumor necrosis factorα (TNF-α), β-defensin2, and interleukins (IL-1β, IL-6, IL-8) were performed on RNA extracted from endometrial tissue and peripheral white blood cells (WBCs) using quantitative real-time PCR. Endometrial tissue was also used for histopathology and cytology. In experimental goats, the mRNA expression of TLR4 and proinflammatory cytokines were increased within 24 h pi (P < 0.01) in endometrium and WBCs. Similarly, expression of β-defensin2 was higher at 72 h pi in endometrium (P < 0.001), and at 120 h pi in WBCs (P < 0.05). The %PMNs in the experimental group increased up to 92.16 ± 3.95% at 3 h pi (P < 0.001). Endometrial histopathology revealed a severe inflammatory response at 3 to 12 h pi, whereas no changes were detected in the control group. In conclusion, intrauterine infusion of E. coli in goats resulted in a rapid activation of the local innate immune response, characterized by infiltration of PMNs into the endometrium and up-regulation of gene expression for TLR4, cytokines and β-defensin2.     

Neuropeptide Y reduces the expression of <Emphasis Type="Italic">PLCB2</Emphasis>, <Emphasis Type="Italic">PLCD1</Emphasis> and selected <Emphasis Type="Italic">PLC</Emphasis> genes in cultured human endothelial cells

The ability of mycobacteria to grow and invade target tissues is the key component in the process of Mycobacterium bovis infection. Therefore, analysis of the proteins responsible for cell invasion will assist clinicians in combating bovine tuberculosis. The Mb1514 gene of M. bovis encodes a hypothetical invasion protein (designated here as MbINV protein), whose function has not yet been directly identified. In this study, the Mb1514 gene from M. bovis was cloned, and expressed in E. coli. The recombinant MbINV protein (a single band of approximately 28 kDa) was purified for biological analysis. Our data demonstrated that recombinant MbINV protein significantly inhibited the viability of RAW264.7 macrophages in a dose-dependent manner (P < 0.05), and induced cell necrosis, indicating that the protein is toxic. MbINV protein infection significantly enhanced the mRNA expression levels of TNF-α, IL-1β, and NOS2 (P < 0.01), suggesting that MbINV protein may be one of the virulence factors which directly interact with macrophages and modulate the host immune response to M. bovis. An invasion inhibition assay showed that MbINV-inhibited M. bovis invasion of RAW264.7 cells in a concentration-dependant manner, demonstrating it is an invasion protein.     

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) stimulates IL-10 production via the PI3K/Akt and p38 MAPK pathways in human lung epithelial cells

Infection of human cells with mycobacteria has been shown to result in the production of anti-inflammatory cytokines. However, the signaling pathways that regulate the Mycobacterium bovis BCG-induced interleukin (IL)-10 production are currently unknown. In the present study, we investigated the involvement of phosphatidylinoditol 3-kinase (PI3K)/Akt and the p38 MAPK signaling pathways in the secretion of IL-10 in human lung epithelial cells (A549) after infection with M. bovis BCG. Treatment of A549 cells with LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and wortmannin, two PI3K inhibitors, inhibited M. bovis BCG-induced IL-10 production. Stimulation of cells with M. bovis BCG caused an increase in Akt phosphorylation in a time-dependent manner, which was inhibited by wortmannin. In addition, treatment of A549 cells with an Akt inhibitor significantly blocked M. bovis BCG-induced IL-10 production. Moreover, the p38 inhibitor SB203580 significantly decreased IL-10 production in a dose-dependent manner, whereas M. bovis BCG-induced IL-10 secretion was completely unaffected by the MEK inhibitor PD98059. Finally, the inhibition of PI3K did not significantly affect p38 MAPK activation in M. bovis BCG-infected cells, indicating that PI3K activity is not required for the M. bovis BCG-induced phosphorylation of p38 MAPK. Collectively, these data suggest that the PI3K/Akt and p38 MAPK signaling pathways play an important role in the regulation of M. bovis BCG-induced IL-10 secretion in A549 cells.     

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.     

Endometrial expression of selected transcripts involved in prostaglandin synthesis in cows with endometritis

Several cytokines and prostaglandins play an important role in preparing the endometrium for implantation and mediating pro-inflammatory events. The aim of the present study was to examine mRNA expression of interleukin 1α (IL-1α), interleukin receptor antagonist (IL-1-RN), cytosolic prostaglandin E synthase (cPGES), microsomal PGES (mPGES-1 and mPGES-2) and lipocalin-type PGDS (L-PGDS) in the bovine endometrium. Endometrial epithelium samples were collected ex vivo from cows with different status of health at day 21-27 postpartum on a dairy farm. Three groups (n = 9 animals each) were defined: (1) healthy cows with no signs of endometritis (control group), (2) cows with subclinical endometritis, and (3) cows with signs of clinical endometritis. Oestrous cycle-dependent mRNA expression pattern was investigated using bovine endometrial epithelial cells from healthy uteri collected at the abattoir. These uteri were classified into post-ovulatory, early-to-mid luteal, late luteal or pre-ovulatory phase (n = 8 animals for each cycle phase). After collecting endometrial epithelium using the cytobrush-method, mRNA analysis was performed by real-time RT-PCR. L-PGDS, IL-1α and IL-1-RN mRNA were expressed significantly higher (P < 0.05) in the endometrium of cows with subclinical or clinical endometritis compared with healthy cows. A twofold lower cPGES mRNA expression (P < 0.05) was detected in cows with subclinical endometritis compared to healthy cows. L-PGDS and IL-1-RN mRNA expression was increased (P < 0.05) after ovulation compared with the pre-ovulatory or luteal phase, respectively. These results support the hypothesis that a dys-regulated cytokine and/or prostaglandin profile in the uterus could be induced by subclinical endometritis or clinical endometritis.     

Molecular cloning and endometrial expression of porcine amphiregulin

The porcine amphiregulin gene was previously reported to be within the quantitative trait locus (QTL) for uterine capacity on chromosome 8. Because amphiregulin stimulates cell proliferation, the amphiregulin gene might be responsible for this QTL. The objectives of this study were to clone amphiregulin cDNA and compare endometrial expression of its mRNA in pregnant Meishan (M) and White composite (WC) pigs. We obtained two amphiregulin cDNAs, one with 1,221 bp and another with 1,109 bp. The 112 bp difference corresponded to exon 5 of the human amphiregulin gene, which codes for the cytoplasmic domain. Endometrial mRNA expression of amphiregulin was significantly lower in M pigs than in WC pigs during early pregnancy (day 15 - 40 of gestation). Amphiregulin mRNA expression in the endometrium of both M and WC pigs increased (P < 0.01) from days 15 to 20, decreased (P = 0.01) from days 20 to 30, and did not change between days 30 and 40. This may result in reduced amphiregulin protein production leading to the slower development of M conceptuses, contributing to greater uterine capacity and litter size in prolific Chinese M pigs. Porcine genomic sequences isolated from a bacterial artificial chromosome genomic library contained exon 5, suggesting that the deletion of exon 5 in the mRNA may be due to differential splicing. The amphiregulin gene consisted of six exons and five introns spanning 10.3 kb. Mol.     

Vaccination with a BCG Strain Overexpressing Ag85B Protects Cattle against Mycobacterium bovis Challenge

Mycobacterium bovis is the causative agent of tuberculosis in cattle but also infects other animals, including humans. Previous studies in cattle have demonstrated that the protection induced by BCG is not complete. In order to improve the protection efficacy of BCG, in this study we overexpressed Ag85B in a BCG Pasteur strain, by using an expression system based on the use of an auxotrophic strain for the leucine amino acid, and complementation with leuD. We found that vaccination of cattle with BCG overexpressing Ag85B induced higher production of IL-17 and IL-4 mRNA upon purified protein derivative (PPDB) stimulation of peripheral blood mononuclear cells (PBMCs) than vaccination with BCG. Moreover, the IL-17 mRNA expression after vaccination negatively correlated with disease severity resulting from a subsequent challenge with M. bovis, suggesting that this cytokine is a potential biomarker of cattle protection against bovine tuberculosis. Importantly, vaccination with the recombinant BCG vaccine protected cattle better than the wild-type BCG Pasteur.     

Effects of matrix proteins on the expression of matrix metalloproteinase-2, -9, and -14 and tissue inhibitors of metalloproteinases in human cytotrophoblast cells during the first trimester.

The activity of matrix metalloproteinases (MMPs) specifies the ability of the trophoblast cell to degrade extracellular matrix (ECM) substrates. Usually the process of normal human placentation involves a coordinated interaction between the fetal-derived trophoblast cells and their microenvironment in the uterus. In this study, the effects of ECM proteins on the expression of MMP-2, -9, and -14 (membrane-type MMP-1); and the production of tissue inhibitors of metalloproteinase (TIMP) types -1, -2, and -3 have been investigated. Cytotrophoblast cells at 9 or 10 wk of gestation were cultured on various ECM coated dishes under serum-free conditions. Gelatin zymography analysis showed that cells grown on fibronectin (FN), laminin (LN), and vitronectin (VN) secreted more MMP-9 (about 1.5- to 3-fold more) than cells cultured on collagen I (Col I), whereas the secretion of MMP-9 by cells cultured on collagen IV (Col IV) was only half that by the cells on Col I. Northern Blot analysis gave the same results as zymography, indicating that expression of the MMP-9 gene in cytotrophoblast cells can be affected by matrix proteins. There was no significant difference in the expression of MMP-2 either at protein or mRNA levels among the cells cultured on the different matrix substrates. The expression of MMP-14 was regulated in a manner similar to that of MMP-2. Using ELISA, we detected higher levels of TIMP-1 in the culture medium of cells grown on VN, LN, and FN compared with that grown on Col I. But the expression of TIMP-3 mRNA was remarkably inhibited by VN, and ECM proteins had no effect on TIMP-1 and TIMP-2 mRNA expression. It was also observed that cultured cytotrophoblast cells expressed the corresponding receptors for the tested matrix proteins, such as integrins alpha(1), alpha(5), alpha(6), beta(1), and beta(4). Furthermore, the adhesiveness of cytotrophoblast cells on Col I, Col IV, FN, and LN was increased by 62%, 45%, 21%, and 22%, respectively, when compared with adhesiveness on VN. Isolated cytotrophoblast cells remained stationary when cultured on dishes coated with Col I and Col IV, but they assumed a more motile morphology and aggregated into a network when cultured on LN and VN. These data indicate that human trophoblast cells interact with their microenvironment to control their behavior and function.     

Correction of the defective extracellular matrix of Ehlers-Danlos syndrome skin fibroblasts by dexamethasone.

Skin fibroblasts derived from Ehlers-Danlos syndrome (EDS) patients lack an organized extracellular matrix (ECM) of fibronectin (FN) and often show an accumulation of cytoplasmic FN. The treatment of EDS cells of different types (I to VIII) with 10-⁷ M dexamethasone (dex), as well as cocultivation with control fibroblasts, induced in most cases the assembly of a FN-like ECM. The study of FN mRNA expression by dot-blot hybridization and of FN released into the culture media of EDS cells showed that the correction of the defective FN-ECM of EDS cells by dex treatment is associated in most cases with an increase of FN mRNA synthesis and of secreted FN.     

Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin

Given prior evidence that adhesion molecules play critical roles in T cell recognition, it is important to identify new adhesion pathways and explore their role in T cell activation. Our studies of T cell proliferation complement concurrent studies of T cell adhesion; both demonstrate that resting CD4+ human T lymphocytes express the VLA integrins VLA-4, VLA-5, and VLA-6, and can use these receptors to interact with the extracellular matrix (ECM) proteins fibronectin (VLA-4 and VLA-5) and laminin (VLA-6). VLA-dependent interaction of resting human CD4+ T cells with fibronectin (FN) and laminin (LN) facilitates CD3-mediated T cell proliferation. Specifically, T cells do not proliferate in response to a wide range of concentrations of a CD3 mAb, OKT3, immobilized on plastic. However, coimmobilization with the CD3 mAb of FN or LN, but not other ECM proteins such as fibrinogen and collagen, consistently results in strong T cell proliferation. mAb blocking studies demonstrate that three VLA integrin receptor/ligand interactions mediate costimulation: VLA-4/FN, VLA-5/FN, and VLA-6/LN. VLA-5-dependent binding to FN but not costimulation by FN can be specifically blocked with peptides containing the RGD (arg-gly-asp) tripeptide sequence whereas VLA-4-dependent binding and costimulation can both be efficiently inhibited by a 12 amino acid peptide, LHGPEILDVPST (leu-his-gly-pro-glu-iso-leu-asp-val-pro-ser-thr), derived from the alternatively spliced IIICS region of FN. The costimulation provided by FN and LN in this system is stronger than and distinct from costimulatory signals provided by cytokines, such as IL-1 beta, IL-6,, and IL-7. These results suggest that, such as other adhesion molecules, T cell VLA integrins may also function in a dual capacity as adhesion and signalling molecules. In addition, they suggest that the interaction of T cells in vivo with ECM via VLA integrins plays a role not only in T cell migratory processes but may also influence Ag-specific T cell recognition.     

TWEAK appears as a modulator of endometrial IL-18 related cytotoxic activity of uterine natural killers

Background

TWEAK (Tumor necrosis factor like WEAK inducer of apoptosis) is highly expressed by different immune cells and triggers multiple cellular responses, including control of angiogenesis. Our objective was to investigate its role in the human endometrium during the implantation window, using an ex-vivo endometrial microhistoculture model. Indeed, previous results suggested that basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity.

Methodology/Principal Findings

Endometrial biopsies were performed 7 to 9 days after the ovulation surge of women in monitored natural cycles. Biopsies were cut in micro-pieces and cultured on collagen sponge with appropriate medium. Morphology, functionality and cell death were analysed at different time of the culture. We used this ex vivo model to study mRNA expressions of NKp46 (a uNK cytotoxic receptor) and TGF-beta1 (protein which regulates uNK cytokine production) after adjunction of excess of recombinant IL-18 and either recombinant TWEAK or its antibody. NKp46 protein expression was also detailed by immunohistochemistry in selected patients with high basic mRNA level of IL-18 and either low or high mRNA level of TWEAK. The NKp46 immunostaining was stronger in patients with an IL-18 over-expression and a low TWEAK expression, when compared with patients with both IL-18 and TWEAK high expressions. We did not observe any difference for TWEAK expression when recombinant protein IL-18 or its antibody was added, or conversely, for IL-18 expression when TWEAK or its antibody was added in the culture medium. In a pro-inflammatory environment (obtained by an excess of IL-18), inhibition of TWEAK was able to increase significantly NKp46 and TGF-beta1 mRNA expressions.

Conclusions/Significance

TWEAK doesn''t act on IL-18 expression but seems to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed. Thus, TWEAK appears as a crucial physiological modulator to prevent endometrial uNK cytotoxicity in human.     

Regulation of thrombospondin-1 expression in alternatively activated macrophages and adipocytes: role of cellular cross talk and omega-3 fatty acids

Thrombospondin-1 (TSP-1) expression in human adipose positively correlates with body mass index and may contribute to adipose dysfunction by activating transforming growth factor-β and/or inhibiting angiogenesis. Our objective was to determine how TSP-1 is regulated in adipocytes and polarized macrophages using a coculture system and to determine whether fatty acids, including the ω-3 fatty acid docosahexaenoic acid (DHA), regulate TSP-1 expression. Coculture of M1, M2a or M2c macrophages with adipocytes induced TSP-1 gene expression in adipocytes (from 2.4- to 4.2-fold, P<.05), and adipocyte coculture induced TSP-1 gene expression in M1 and M2c macrophages (M1: 8.6-fold, M2c: 26-fold; P<.05). TSP-1 protein levels in the shared media of adipocytes and M2c cells were also strongly induced by coculture (>10-fold, P<.05). DHA treatment during the coculture of adipocytes and M2c macrophages potently inhibited the M2c macrophage TSP-1 mRNA level (97% inhibition, P<.05). Adipocyte coculture induced interleukin (IL)-10 expression in M2c macrophages (10.1-fold, P<.05), and this increase in IL-10 mRNA expression was almost completely blocked with DHA treatment (96% inhibition, P<.05); thus, IL-10 expression closely paralleled TSP-1 expression. Since IL-10 has been shown to regulate TSP-1 in other cell types, we reduced IL-10 expression with siRNA in the M2c cells and found that this caused TSP-1 to be reduced in response to adipocyte coculture by 60% (P<.05), suggesting that IL-10 regulates TSP-1 expression in M2c macrophages. These results suggest that supplementation with dietary ω-3 fatty acids could potentially be beneficial to adipose tissue in obesity by reducing TSP-1 and fibrosis.     

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.     

Glucose Availability and AMP-Activated Protein Kinase Link Energy Metabolism and Innate Immunity in the Bovine Endometrium

Defences against the bacteria that usually infect the endometrium of postpartum cattle are impaired when there is metabolic energy stress, leading to endometritis and infertility. The endometrial response to bacteria depends on innate immunity, with recognition of pathogen-associated molecular patterns stimulating inflammation, characterised by secretion of interleukin (IL)-1β, IL-6 and IL-8. How metabolic stress impacts tissue responses to pathogens is unclear, but integration of energy metabolism and innate immunity means that stressing one system might affect the other. Here we tested the hypothesis that homeostatic pathways integrate energy metabolism and innate immunity in bovine endometrial tissue. Glucose deprivation reduced the secretion of IL-1β, IL-6 and IL-8 from ex vivo organ cultures of bovine endometrium challenged with the pathogen-associated molecular patterns lipopolysaccharide and bacterial lipopeptide. Endometrial inflammatory responses to lipopolysaccharide were also reduced by small molecules that activate or inhibit the intracellular sensor of energy, AMP-activated protein kinase (AMPK). However, inhibition of mammalian target of rapamycin, which is a more global metabolic sensor than AMPK, had little effect on inflammation. Similarly, endometrial inflammatory responses to lipopolysaccharide were not affected by insulin-like growth factor-1, which is an endocrine regulator of metabolism. Interestingly, the inflammatory responses to lipopolysaccharide increased endometrial glucose consumption and induced the Warburg effect, which could exacerbate deficits in glucose availability in the tissue. In conclusion, metabolic energy stress perturbed inflammatory responses to pathogen-associated molecular patterns in bovine endometrial tissue, and the most fundamental regulators of cellular energy, glucose availability and AMPK, had the greatest impact on innate immunity.     

CXCR4, regulated by HIF1A,promotes endometrial breakdown via CD45+ leukocyte recruitment in a mouse model of menstruation

Menstruation is a specific physiological phenomenon in female humans that is regulated by complex molecular mechanisms. However, the molecular network involved in menstruation remains incompletely understood. Previous studies have suggested that C-X-C chemokine receptor 4 (CXCR4) is involved; however, how CXCR4 participates in endometrial breakdown remains unclear, as do its regulatory mechanisms. This study aimed to clarify the role of CXCR4 in endometrial breakdown and its regulation by hypoxia-inducible factor-1 alpha (HIF1A). We first confirmed that CXCR4 and HIF1A protein levels were significantly increased during the menstrual phase compared with the late secretory phase using immunohistochemistry. In our mouse model of menstruation, real-time PCR, western blotting, and immunohistochemistry showed that CXCR4 mRNA and protein expression levels gradually increased from 0 to 24 h after progesterone withdrawal during endometrial breakdown. HIF1A mRNA and HIF1A nuclear protein levels significantly increased and peaked at 12 h after progesterone withdrawal. Endometrial breakdown was significantly suppressed by the CXCR4 inhibitor AMD3100 and the HIF1A inhibitor 2-methoxyestradiol in our mouse model, and HIF1A inhibition also suppressed CXCR4 mRNA and protein expression. In vitro studies using human decidual stromal cells showed that CXCR4 and HIF1A mRNA expression levels were increased by progesterone withdrawal and that HIF1A knockdown significantly suppressed the elevation in CXCR4 mRNA expression. CD45⁺ leukocyte recruitment during endometrial breakdown was suppressed by both AMD3100 and 2-methoxyestradiol in our mouse model. Taken together, our preliminary findings suggest that endometrial CXCR4 expression is regulated by HIF1A during menstruation and may promote endometrial breakdown, potentially via leukocyte recruitment.     

Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit

The progesterone receptor (PR) plays a pivotal role in the maturation process of the secretory endometrium, implantation and maintenance of pregnancy in rabbits. To determine the dynamics of PR gene expression and its physiological significance, the endometrial expression of PR and PR mRNA were evaluated and compared with the expression of the progesterone-regulated uteroglobin (UG) gene during 0–5 days post-coitus in rabbits. The results of immunoblot experiments indicated the presence of PR in endometrial cell extracts from days 1–4 of pregnancy with maximum PR immunostaining on day 2, followed by a marked diminution until its complete disappearance on day 5. When endometrial PR mRNA content was assessed by Northern blots, the results were similar to those of PR immunostaining, with maximal concentrations on the second day after mating. However, PR mRNA levels were still high on day 3, despite the concomitant decrease in immunostainable PR. Endometrial UG gene expression, on the other hand, exhibited a different time sequence. Thus, the UG content in uterine flushings progressively increased from day 3 after mating, reaching maximal levels on the fifth day. The endometrial UG mRNA content presented a similar profile, as its maximum concentration occurred on days 4–5. The overall results indicate that endometrial PR is down-regulated at both the mRNA and protein levels, possibly by endogenous progesterone during early pregnancy. The striking observation that maximal expression of endometrial UG gene products occurred when PR and its mRNA are no longer detectable suggests an important role for this progesterone-binding uterine protein during the preimplantation period. © 1993 Wiley-Liss, Inc.     

Adhesion of human monocytic THP-1 cells to endothelial cell adhesion molecules or extracellular matrix proteins via beta1 integrins regulates heparin binding epidermal growth factor-like growth factor (HB-EGF) expression

Adherence to endothelium and then to the extracellular matrix is a prerequisite for extravasation of monocytes into injured tissues. There, monocytes differentiate into macrophages and express heparin binding epidermal growth factor-like growth factor (HB-EGF), a key growth factor involved in normal wound healing. We investigated whether the interaction of human monocytic THP-1 cells with the endothelial cell adhesion molecules (vascular CAM-1, VCAM-1; intercellular adhesion molecule-1 ICAM-1 and endothelial-selectin, E-selectin), or the extracellular matrix (ECM) proteins (fibronectin, FN; laminin, LN and fibrinogen, FG) regulate HB-EGF expression. We have shown that adherence of THP-1 cells via VCAM-1, E-selectin or FN, which are all overexpressed at sites of inflammation, potentiates HB-EGF mRNA expression. In contrast, adhesion of THP-1 cells via ICAM-1 or FG, has no significant effect. Since THP-1 cells interact with ICAM-1 and FG through beta2 integrins, and with VCAM-1 and FN via beta1 integrins, regulation of HB-EGF expression appears to be specific to beta1 integrin ligation. In addition, we demonstrate that THP-1 binding to LN, through the beta1 integrin VLA-6, down regulates HB-EGF expression. Thus physiologically, transient destruction of LN and expression of VCAM-1, E-selectin and fibronectin at sites of inflammation, may locally induce HB-EGF overexpression.