Human trophoblast adhesion to matrix proteins: inhibition and signal transduction

At the time of implantation, the extracellular matrix proteinslaminin and fibronectin are abundant in the decidua and aredistributed pericellularly around each individual stromal cell.First trimester human trophoblast expresses both laminin andfibronectin receptors, specifically the ₁ß_{1 5}ß₁₆ß₁ and ₆ß₄ integrin heterodimers. In thisstudy we have demonstrated that in-vitro adhesion of first trimesterhuman trophoblast to purified extracellular matrix proteinsand to purified decidual stromal cell monolayers can be inhibitedby monoclonal antibodies directed against appropriate integrinsubunits and by synthetic peptides containing an arginine-glycine-asparticacid sequence. Monoclonal antibodies (mAbs) to the ₅ and ß₁integrin subunits and a synthetic peptide significantly inhibitedadhesion to fibronectin. Binding of trophoblast to laminin wasblocked with mAbs to the ₆ and ß₁ but not ₁ and ß₄integrinsubunits. Similarly, integrin-mediated adhesion to monolayersof decidual stromal cells could be blocked with mAbs to the₅, ₆, ß₆ and ß₄ integrin subunits. Integrin-mediatedsignal transduction in normal and malignant trophoblast wasinvestigated by Western blotting. A 115 kDa protein was themajor tyrosine phosphorylated protein detected in trophoblastafter binding to laminin or fibronectin. The profile of tyrosinephosphorylated proteins differed for malignant trophoblast. integrins/matrix/signal transduction/trophoblast     

Up-regulation of matrix metalloproteinase-9 in human myometrium during labour: a cytokine-mediated process in uterine smooth muscle cells

The pregnant uterus undergoes dramatic changes of tissue remodellingduring the labour and post-partum period. We studied the productionof matrix metalloproteinase 9 (MMP-9), as a major contributorof tissue remodelling, in human myometrium at parturition. Theregulation of proMMP-9 by interleukin-1ß (IL-1ß)and tumour necrosis factor- (TNF-) was also investigated inhuman myometrial smooth muscle cells. MMP-9 was present in myometrialsmooth muscle cells, interstitial fibroblasts and inflammatorycells. The gelatinolytic activities of proMMP-9 in myometriumincreased dramatically during labour. IL-1ß and TNF- inducedproMMP-9 in myometrial smooth muscle cells, but these effectsdid not seem to be mediated by protein kinase C. On the otherhand, neither 17ß-oestradiol nor progesterone itself affectedproMMP-9 production in myometrial smooth muscle cells. Moreover,progesterone, which is known as a physiological suppressor ofMMP-9 in other species, did not decrease the IL-1ß- andTNF--induced production of proMMP-9. These results suggest thatIL-1ß and TNF- are effective up-regulators of proMMP-9in the tissue remodelling of human myometrium during labour. interleukin-1ß/matrix metalloproteinase-9/myometrium/smooth muscle cell/tumour necrosis factor- Notes ¹ To whom correspondence should be addressed     

A positive correlation between expression of {beta}1-integrin cell adhesion molecules and fertilizing ability of human spermatozoa in vitro

The purpose of this study was to investigate firstly whetherß1-integrin cell adhesion molecules are expressedby human spermatozoa, and secondly whether there is any relationshipbetween the expression of ß1-integrin cell adhesionmolecules and the fertilizing ability of human spermatozoa invitro. A total of 50 semen samples were examined. The sampleswere obtained from the male partners of couples undergoing in-vitrofertilization (IVF) for either unexplained, tubal or male factorinfertility. A panel of six monoclonal antibodies against p1-integrincell adhesion molecules and immunohistochemical techniques wereused to identify the presence of these molecules on the spermatozoa.The percentage of spermatozoa showing strong immunolabellingwith each monoclonal antibody was assessed in each sample. Therelationship between these results and the aetiology of infertilityand incidence of fertilization was examined. ß1-lntegrins,and primarily the ones with 4-, 5- and 6-chains, were expressedby human spermatozoa. Compared with semen samples from unexplainedor male factor infertility patients, samples from tubal infertilitypatients had a significantly higher (P < 0.05) percentageof spermatozoa expressing adhesion molecules. There was a positivecorrelation between the expression of 4, 5 and 6 adhesion moleculesand the fertilizing ability of spermatozoa. The positive correlationbetween the presence of certain ß1-integrin cell adhesionmolecules and the fertilizing ability of human spermatozoa suggeststhat integrins may be putative determinants in egg—spermrecognition and interaction. fertile/infertile men/integrins/spermatozoa     

Cell adhesion molecules on the oocyte and preimplantation human embryo

The presence of cell adhesion molecules on human oocytes, earlyembryos, and pre-hatched blastocysts was examined by indirectimmunofluorescence and compared to the distribution found onfirst trimester villous placenta with the same antibodies. Sixintegrin subunits (3, V, ß1, ß3, ß4,ß5) were observed consistently throughout preimplantationdevelopment. Evidence was also obtained for the presence ofintegrin subunits 2, 4, L, ß2, and ß7 ona small number of oocytes. A more restricted developmental analysisof E-cadherin, ICAM-1, NCAM, and VCAM-1 demonstrated that thesecell adhesion molecules are also present on oocytes and earlyembryos. L-selectin was detected on oocytes but was not foundon 8-cell embryos. The oocyte and early blastomeres have complexsurfaces in which the integrin and CAM families are represented. cadherin/cell adhesion molecules/human embryo/immunofluorescence/oocyte     

Gelatinase and oncofetal fibronectin secretion is dependent on integrin expression on human cytotrophoblasts

Collagenolytic activity of cytotrophoblasts is stimulated byglycoproteins of the extracellular matrix and since this stimulationcan possibly occur through integrins, we measured the gelatinolyticactivity of villous and extravillous cytotrophoblasts accordingto the type of integrins expressed on these cells. Cytotrophoblastswere isolated from legal abortions, immunopurified with anti-CD45,separated according to their expression of histocompatibiltty-linkedantigen (HLA)-G, ₆ or ₅ integrin subunits and cultured for 5days on plastic or agarose. Fetal fibronectin, human chorionicgonadotrophin (HCG) and the gelatinolytic activity were measuredin the culture supernatants. Following immunopurrfication withanti-CD45, the gelatinolytic activity of cytotrophoblasts wassignificantly higher than before, indicating that contaminatinglymphomyeloid cells secreted gelatinolytic inhibitors. HLA-Gpositive cells secreted significantly more gelatinases thanHLA-G negative cells but their HCG secretion was similar. Comparedto ₅ positive cells, ₆ positive cytotrophoblasts secreted significantlymore gelatinases, significantly less fibronectin but similaramounts of HCG. We conclude that during trophoblast invasion,extravillous cytotrophoblasts (HLA-G positive) expressing the₆ integrin subunit represent the invasive population of cells(high gelatinase and low fibronectin secretion). When expressionof the ₅ integrin subunit is turned on, their invasive behaviourceases and they secrete low amounts of gelatinases and highconcentrations of fibronectin. fibronectin/gelatinase/integrins/trophoblast invasion ^*Part of the results described here were presented at the ESHREmeeting in Brussels in June 1994.     

Tumour necrosis factor-{alpha}-mediated dyscohesion of epithelial cells is associated with disordered expression of cadherin/{beta}-catenin and disassembly of actin filaments

Tumour necrosis factor (TNF)- induced, in a time- and dose-dependentfashion, dyscohesion (cell-cell dissociation) of the endometrialepithelial cells. TNF- impaired the ability of cells to aggregateand to attain compaction. The cell-cell adherent junction isa specialized region of the plasma membrane where cadherin moleculesact as adhesion molecules and actin filaments are densely associatedwith the plasma membrane through a well-developed plasmalemmalundercoat. Dyscohesion induced by TNF- was associated with thedisordered expression of cadherin/ß-catenin at thesites of cell-cell contact. In addition, within the time-framethat dyscohesion was induced, TNF- down-regulated the expressionof actin mRNA only at 100 ng/ml without modulating the overallamount of actin protein, its ß-isoform or the amountof ribosylated actin. However, TNF--mediated dyscohesion ofepithelial cells was associated with loss of plasmalemmal undercoatas well as intracytoplasmic aggregates of F-actin and a simultaneousincrease in G-actin. The effect of cytochalasin-B, which disruptsactin filaments on cell-cell binding, was less pronounced thanthe effect of TNF-, suggesting that the effect of this cytokineon dyscohesion is not solely dependent on the disassembly ofactin filaments. These findings show that the induction of disorderedexpression of adhesion molecules, as well as disassembly ofactin filaments, are implicated in the dyscohesion induced byTNF-. actin/cell adhesion/cytochalasin/epithelial cells/TNF-     

Implantation: Impact of trophoblast penetration through the basal membrane on the efficacy of drug therapy in tubal pregnancies

Concentrations of -human chorionic gonadotrophin (HCG) of 2500IU/l are generally considered to be maximal for successful drugtherapy of tubal pregnancies [instillation of prostaglandin-F₂(PGF₂) or hyperosmolar glucose]. The purpose of our study wasto ascertain if there was an association between the significantlyhigher failure rates above this threshold value and the histologicallydetermined anatomopathological substratum. We therefore evaluatedthe impact of trophoblast penetration through the basal membraneof the Fallopian tube on the efficacy of drug therapy. Pre-operativeserum -HCG concentrations were compared with the histologicallydetermined trophoblast penetration, distinguishing between ectopicpregnancies with intra-luminal growths up to the myosalpinx,and those with extra-luminal growths going beyond the basalmembrane and penetrating the myosalpinx. Basic data were obtainedfrom a group of patients who received primary surgical treatmentbut it had never been the intention for them to receive drugtherapy (independently of their initial -HCG values; group I,n = 43). These reference data were compared with the findingsin preparations from another group of patients obtained duringsecondary surgical intervention, performed to achieve finalcure of tubal pregnancy after failure of primary PGF₂ instillation(group II, n = 30). Group I patients showed a significantlyhigher rate of intra-luminal trophoblast growths (P = 0.0001)at -HCG values <2500 IU/l; above this threshold value, extra-luminalspread was found significantly more often (P = 0.0001). In histologicalpreparations from group II, however, the number of extra-luminalgrowths was significantly higher even at low -HCG values (P= 0.007); at values above the threshold level, the distributionsin the two groups were similar. These results suggest that drugtherapy of tubal pregnancy becomes inefficient in tubal pregnanciesas soon as the trophoblast penetrates the basal membrane ofthe Fallopian tube.     

Expression and function of fibronectin binding integrins on rat mast cells

Adhesion molecules of the integrin family are implicated notonly in leukocyte migration but also in leukocyte activation.Here we characterize the expression and function of fibronectinreceptor integrins on rat mast cells. A rat basophilic leukemiacell line (RBL-2H3) and phorbol esterstimulated rat peritonealmast cells adhered to fibronectin (FN), vitronectin and fibrinogen.These mast cells expressed fibronectin receptor integrins, Includingvery late antigen (VLA)-4, VLA-5 and vitronectin receptor (VNR),as estimated by immunofluorescent staining and inhibition ofFN adherence by newly established mAbs reactive with the rat4 (MR4-1), 5 (HM5-1) or ß3 (HMß3-1) chainsof the integrin molecules. The ß-hexosaminidase release,a marker for mast cell degranulation, triggered by high affinityIgE receptor (FcRI)-medlated stimulation, was enhanced by adhesionof RBL-2H3 cells to either immobilized FN, MR4-1, HM5-1 or HMß3-1.This FN enhancement of ß-hexosaminidase release wasinhibited by soluble MR4-1, HM5-1 and HMß3-1 as wellas by GRGDSP and DELPQLVTLPHPNHLGPEILDVPST peptides which abrogateVLA-5/VNR and VLA-4 binding to FN respectively. In vivo, passivecutaneous anaphylaxis induced by IgE anti-DNP and DNP-BSA wasinhibited by concurrent s.c. injection of MR4-1, HM5-1 and HMß3-1.These results demonstrate that FN receptor integrins expressedon rat mast cells play an important role in regulating mastcell activation both in vitro and in vivo.     

Signal transduction in human B cells initiated via Ig{beta} ligation

Ig and Igß heterodimers are non-covalently associatedwith Ig to compose the antigen receptor complexes on B cells.The demonstration that different sets of tyrosine kinases bindto the cytoplasmic tails of Ig and Igß suggests thatIg and Igß may activate distinct second messengerpathways. In this study, we examined the effects of mAbs againstan exposed epitope of human Igß on pre-B and B celltriggering. Cross-linkage of Igß on B cells leadsto activation of tyrosine kinases, hydrolysis of phosphatidylinositides,and elevation of intracellular Ca²⁺, effects qualitatively identicalto those of anti-_µ mAbs. Our observations thus indicatethat cross-linking of Igß does not segregate signaltransduction pathways connected with the cytoplasmic talls ofIg and Igß. Ig ligation has been reported to be moreeffective in triggering pre-B than B cells, whereas our resultsindicated that Igß ligation is more efficient in triggeringB than pre-B cells. In addition to their activation properties,the anti-Igß mAbs effectively modulated B cell receptorcomplexes and blocked terminal differentiation of all plasmacell isotypes. The findings support the idea that anti-Igßcould serve as a universal B cell immunosuppressant.     

HLA-DR and H-2E transgenes differentially mediate TCR-specific positive selection

The use of HLA transgenic mice in models of immunity and diseaseassumes that human MHC molecules are able to contribute towardthe positive selection of the mouse TCR repertoire. As an initialstep towards analysis of this we have compared the relativeability of DR/Eß or E/Eß complexes to induceT cell receptor (TCR) positive selection in H-2Ea and HLA-DRAtransgenic mice lacking endogenous E. The results show that,like E/Eß, the hybrid DR/ß complexes arecapable of mediating positive selection of V_ß2⁺;,V_ß6⁺, and V_ß10⁺ cells. However, differenceswere found between the effects of the two transgenes. Thus,while V_ß6⁺ cells were efficiently selected in bothH-2Ea and DRA transgenic mice, positive selection of V_ß10⁺cells was less apparent in the DRA transgenic mice. Variationbetween Ea and DRA transgenic mice is consistent with the notionthat this process is dependent on differential binding of endogenouspeptides to the E/Eß and DR/Eß complexes.Furthermore, contrary to expectations, in neither set of micewas positive selection limited solely to the CD4⁺ subset. Thus,examples were found in which V_ß-specific positiveselection was confined to either the CD4⁺ or CD8⁺ subsets, andothers in which both subpopulations were concomltantly increased.In the case of V_ß2 positive selection, H-2Ea transgenicmice showed expansion of these cells in both the CD4⁺ and CD8⁺subpopulations whlle in DRA transgenic mice this occurred predominantlyin the CD8⁺ subpopulatlon.     

Stimulation of prostaglandin (PG) F2{alpha} and PGE2 release by tumour necrosis factor-{alpha} and interleukin-1{alpha} in cultured human luteal phase endometrial cells

Tumour necrosis factor- (TNF-) and interleukin-1 (IL-1) areimportant mediators of cell signalling in the uterus. Prostaglandins(PG) have been implicated in the increase of endometrial vascularpermeability which occurs during the implantation process. Thisstudy evaluates the effect of these two pleiotropic cytokineson PGF₂ and PGE₂ release from human luteal phase endometrialglandular epithelial cells (GEC) and stromal cells (STC) inculture. Basal PGF and PGE release did not differ significantlyfrom each other or among cell types, and declined significantlywith increasing number of days in culture. On day 3, basal PGrelease had decreased to half of that on day 1 of culture. However,both cell types were still able to respond to the addition ofexogenous arachidonic acid (5 µM) on day 3 of culture,with PG release by GEC being elevated 7- to 10-fold and by STCmoderately, but still significantly, on day 4. The permissiveeffect of arachidonic acid on the stimulation of PG releasemay indicate the down-regulation of phospholipase A₂ with continuedtime in culture. However, the addition of arachidonic acid (5µM) on day 0 of culture, while able to cause significantlyincreased PG release from GEC, had no effect on STC. In contrast,the addition of a combination of arachidonic acid (5 µM),and either recombinant human TNF- (10 µg rhTNF-a/l) or10 µg rhlL-1/l, had a synergistic action and caused thesignificantly increased release of PGF and PGE from both celltypes, compared with that achieved with either arachidonic acidor the cytokine alone (although GEC responded more than STC).During the first 24 h after the addition of rhTNF-µ orrhlL-1µ, both cytokines stimulated PG release from bothcell types in a dose- and time-dependent fashion. Neither cycloheximide(10 µM) nor actinomycin D (10 µM) affected basalPG release, but both blocked cytokine-induced PG release fromboth cell types. These results suggest that there is a differentialcontrol of human endometrial cell PG biosynthesis, and thatPG release may be regulated through gene activation. endometrium/human/interleukin-1/prostaglandin/tumour necrosis factor-     

Molecular interactions during pregnancy: Placental mRNA expression of {alpha} and {beta} human chorionic gonadotrophin in early trisomy 18 pregnancies

The placental expression of human chorionic gonadotrophin (HCG)I- and ß-subunits was investigated in eight pregnanciespresenting with trisomy 18 and in 30 normal pregnancies at 11–15weeks gestation. In the control group, the median densitometricscores of placental ß-HCG and I-HCG mRNA were 1.23and 1.74 respectively. In the trisomy 18 group the median ß-HCGmRNA was significantly lower (0.16, Z = 2.29, P<0.05) but     

Human {alpha}{beta} and {gamma}{delta} T cells from unexposed individuals respond to protein antigens of the yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, a dimorphic fungus, causes chronicgranuiomatous mycosis in susceptible individuals. Differentreports have shown that cell-mediated immunity is essentialfor protection against systemic mycosis, including paracoccldloidomycosis.We analyzed the reactivity of ß and T cells fromunexposed Caucasian donors to P. brasiliensis yeast form components.Our results indicate: (I) ß and T cells proliferateafter in vitro stimulation with lysates of P. brasiliensis;(II) similar numbers of ß T cells (f = 1/21,000) andof T cells (f = 1/8000) respond to P. braslllensls; (III) P.braslllensls-reactive T cells express the V9V2 TCR; (Iv) thestimulatory activity of P. brasilensis for both ßand T cells primarily resides in a high molecular weight (100kDa) and in a low molecular weight (<<1 kDa) fraction;(v) the ligands responsible for stimulation of both ßand T cells are sensitive to proteinase treatment We concludethat both ß and T cells from healthy individualsrespond to ubiquitous protein antigens of P. brasiliensis.     

Lymphocytes infiltrating the CNS during inflammation display a distinctive phenotype and bind to VCAM-1 but not to MAdCAM-1

The nature of inflammatory lymphocytes recruited to the CNShas been studied in a model of chronic inflammation. Injectionof killed Corynebacterlum parvum into the cortex of the mousebrain produces a circumscribed inflammatory cellular infiltratearound the injection site, and recruited mononuclear inflammatorycells (IC) can be isolated for flow cytometric analysis. Themajority of IC were T cells. In comparison with the predominantnaive population of mesenteric lymph node T cells, IC T cellsexpress much higher levels of CD44, LFA-1 and ICAM-1, and lowerlevels of CD45RB, features commonly associated with memory (previouslyactivated) cells. In addition, in contrast to the L-selectin⁺6-integrin^low phenotype of naive lymph node T cells, IC T cellslacked L-selectin and were 6-integrin^–. Mac-1, recentlyproposed as another marker of memory T cell differentation,was not displayed by IC T cells, suggesting that Mac-1 expressionmay be heterogeneous among memory T cell subsets. A subset ofmesenteric lymph node (MLN) T cells, probably representing activatedT cells undergoing the naive to memory transition, but not ofIC T cells, expressed high levels of 6-, ß7- and E-integrin.IC and MLN naive T cells expressed comparable levels of 4-integrin,but IC T cells stain poorly with anti-ß7 mAbs andwith mAb DATK 32, specific for the 4ß7 heterodimericlymphocyte homing receptor for the mucosal addressin MAdCAM-1,suggesting that these inflammatory cells express more 4ß1than 4ß7. Consistent with this, in in vitro adhesionassays, brain IC bound better than MLN cells to the 4ß1integrin ligand VCAM-1 and the LFA-1 ligand ICAM-1 but adheredvery poorly to the 4ß7 ligand MAdCAM-1. These findingsare consistent with and extend previous immunohistological studiesof T cells in murine experimental autoimmune encephalomyelitis,and demonstrate a distinctive phenotype for lymphocytes beingpresent in the chronically inflamed brain.     

Morphological and molecular characteristics of living human fetuses between Carnegie stages 7 and 23: immunolocalization of inhibin {alpha} and {beta}a subunits

Transforming growth factor (TGF) is known to have the abilityto modify mitogenic responses of tissues to other peptide growthfactors and therefore may contribute to the rapid growth rateof an embryo. Throughout the TGF superfamily there is a similarfundamental molecular architecture. Included in this superfamilyare inhibin A, activin A and activin B. It has been shown thatactivin is a powerful mesodermal inducing factor in the earlyembryo. The human embryo has shown localization of inhibin inthe gonads after 16 weeks gestation but it has not been previouslyidentified in earlier embryos. The inhibin-activin protein wasfound in a range of tissues including the liver stages 19-21() and stages 19-22 (ß); oesophagus stages 19-22 (and ß); stomach stages 21 and 22 ( and ß);gut stages 16-22 () and 21 and 22 (ß); pericardiumstages 12-22 ( and ß); gonad stages 21 and 22 (ß)stage 22 (); adrenal stages 19-22 ( and ß); urogenitalsystem states 21 and 22 ( and ß); yolk sac stage 12( and ß); mesenchyme stages 16-22 (); surface ectodermstages 13-22 () and stages 16-22 (ßa); notochord stages13-22 (ß) and stages 21 and 22 (); nasal, tracheaand bronchi stages 19-22 ( and ß) leading to speculationof the role of both subunits.     

T cell receptor-triggered activation of intraepithelial lymphocytes in vitro

Intraepithelial lymphocytes (IEL) of the mouse small intestinewere examined for their potential to respond to TCR signallingin vitro. Purified IEL subsets were activated using mAbs specificfor CD3, TCRßor TCR&. Thy-1⁺IEL, regardless ofTCR type, proliferated equally well in response to anti-TCRmAb with or without exogenous IL-2. In contrast, Thy-1^–TCR, CD8^– IEL required exogenous IL-2 for proliferation.No such requirement was observed for Thy-1^– TCR& IELproliferation. IEL proliferation in the absence of added IL-2was due to an IL-2 secretion/IL-2 receptor (IL-2R) autocrinepathway, since mAbs specific for IL-2 and IL-2R inhibited IELproliferation. Thy-1⁺ CD8ß^– CD4⁺CD8⁺ IEL wereunresponsive to TCR-induced proliferation but exhibited highlevels of cytolytic activity upon TCR-triggerlng. Thy-1^–non-cytolytic IEL were induced to express Thy-1 and cytolytlcactivity following activation in vitro. In addition, the involvementof the co-stimulatory molecule CD28 in IEL activation was tested.CD28 was weakly expressed by fresh IEL and anti-CD28 mAb hadno effect on TCR-triggered proliferation. However, anti-TCRstimulation increased CD28 expression on a subset of TCRßIEL and the addition of anti-CD28 mAb resulted in increasedIL-2 production, but not in increased proliferation. Our resultsindicate that IEL, including the purported extrathymlc CD8ß^–subset, can respond to TCR-driven signals via proliferationand/or cytolytlc activity.