Adhesion of carcinoma cells to rat hepatocytes and rat fibronectin is inhibited by the OPAR monoclonal antibody,which is directed against a rat liver-specific carbohydrate epitope

The OPAR mouse monoclonal antibody (mAb) directed against rat hepatocytes was previously shown to inhibit adhesion of TA3/Ha mammary carcinoma cells to hepatocytes. The antigen is abundantly present at the surface of hepatocytes beneath the endothelium of liver capillaries where we have observed invasion of carcinoma cells to occur. The OPAR mAb reacted with three major bands on a Western blot of liver plasma membrane proteins. The same proteins were also seen upon immunoprecipitation from iodinated liver plasma membrane proteins. We have isolated OPAR antigens by lectin wheat germ agglutinin (WGA) and OPAR affinity chromatography. Amino acid sequence analysis revealed that two of the bands were 1-macroglobulin and C4-binding protein, which are serum components produced by hepatocytes. The presence of the epitope on distinct proteins and our previous observation that it can be detected in the Golgi apparatus but not in the endoplasmic reticulum, suggested that OPAR reacts with a liver-specific glycoconjugate. Loss of OPAR reactivity after neuraminidase and N-glycosidase F treatment showed that the epitope contains sialic acid residues on N-linked sugar moieties. OPAR also reacted with rat fibronectin, and inhibited adhesion of TA3/St cells to fibronectin. This explains the inhibition by the OPAR mAb of TA3/St cell adhesion to hepatocytes, which we have shown to be due mainly to interaction with hepatocyte surface-associated fibronectin. However, adhesion of the related TA3/Ha cells to hepatocytes, which is mediated by the ₆P4 integrin, and does not involve binding to fibronectin, is also inhibited. This suggests that ₆₄ on liver-metastasizing carcinoma cells binds to an OPAR epitope-carrying glycoprotein produced by hepatocytes.     

Adhesion to fibronectin via alpha4 integrin (CD49d) protects B cells from apoptosis induced by serum deprivation but not via IgM or Fas/Apo-1 receptors

Apoptosis is a regulated event crucial to the development and proliferation of normal and malignant B cells. We have studied the role of signals delivered via alpha4 integrin on apoptosis triggered by three different pathways on these cells. For apoptosis induced by serum deprivation, culturing B cells on the recombinant fibronectin fragment H89, a known ligand for alpha4beta1 integrin, resulted in statistically significant (P < 0.005) higher viability values (68%, 65% and 67%) for Ramos, Nalm-6 and EHEB cells, respectively, than culturing cells on poly lysine (42%, 42% and 48%). An antialpha4 MoAb reverted the protecting effect, thus confirming that it was due specifically to alpha4 engagement. Similarly, cells cultured on FN-III4-5, a recently identified fibronectin region which binds activated alpha4 integrin, also showed statistically significant higher viability than poly lysine cultures. Alpha4 engagement however, did not prevent apoptosis induced on Ramos cells via surface IgM. Adhesion of IM-9 cells, a myeloma cell line carrying functional Fas receptors, to the H89 fragment neither increased cell viability upon triggering apoptosis via Fas when compared to poly lysine. These results indicate that alpha4 signalling may overcome B cell apoptosis induced by the lack of growth factors but does not seem to affect the IgM or Fas apoptotic pathways, thus suggesting different intracellular mechanisms for these processes.     

Cleavage of integrin by mu-calpain during hypoxia in human endometrial cells

PROBLEM: The distribution and activation of mu-calpain and possible cleavage of integrin in human endometrial cells under hypoxic condition were investigated. METHOD OF STUDY: Human endometrial epithelial and stromal cells were subjected to hypoxia, and subsequently used for immunostaining and western blot analysis. RESULTS: The proform of mu-calpain was detected in the cytoplasm of normal cells, and displayed a substantial decrease after hypoxia. Conversely, the active form of mu-calpain was not detected in normal cells, but was abundant after hypoxia. The cytoplasmic domain of integrin beta3 was also detected in the cytoplasm of endometrial cells. Western blot analysis confirmed that both the proform of mu-calpain and the integrin beta3 cytoplasmic domain decreased during hypoxia. CONCLUSIONS: Mu-calpain is activated in human endometrial cells during hypoxia and that subsequent cleavage of the integrin beta3 cytoplasmic domain may give some adverse effects to the function of human endometrium.     

In vitro regulation of beta1 and beta3 integrin subunits in endometrial epithelial cells from normal endometrium

OBJECTIVE: To evaluate the effect of prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) on integrin expression. DESIGN Cultures of endometrial epithelial cells from normal endometrium. SETTING: All endometrial specimens were obtained from the Obstetrics and gynecology Department of the Catholic University, Rome, Italy. PATIENTS: Four patients were normal menstrual cycles undergoing operative laparoscopy for non-endometrial problems. INTERVENTION: Endometrial samples were collected by uterine courettings. MAIN OUTCOME MEASURE: Immunocytochemistry for beta1 and beta3 integrin subunits. RESULTS: PGE2 clearly enhances both beta1 and beta3 integrin subunit expression. IL-1beta seem to slightly increase only beta3 subunit expression. CONCLUSIONS: In light of the critical role played by eicosanoids in endometrial differentiation, we suggest that PGE2 is also involved in local paracrine regulation of integrin expression.     

Infantile hemangioma is a proliferation of beta 4-negative endothelial cells adjacent to HLA-DR-positive cells with dendritic cell morphology

Although hemangioma is referred as to the most common tumor in infancy, the underlying pathogenetic events and the biologic origin of this benign vascular neoplasm have remained obscure. By using immunohistochemistry on frozen sections of infantile hemangiomas, we show here that proliferating endothelial cells abundantly expressed alpha(v)beta(3) but lacked beta(4) integrins. Instead, regressing and involuting infantile hemangiomas due to treatment with IFN-alpha showed positive staining of beta(4) integrin, which might point to the angiogenic significance of beta(4) integrin in infantile hemangiomas. Moreover, immunofluorescence analysis revealed the existence of HLA-DR(+), mostly CD68(+) and partly DC-SIGN/CD209(+) cells with dendritic cell morphology in the intimate vicinity of hemangiomatous vessels. Such cells were also detected in the dermal microvascular unit in normal skin. The coupled occurrence of vascular structures and perivascular cells that were stained positive with markers of monocyte or macrophage or dendritic cells might suggest that the development of infantile hemangioma is a result of vasculogenesis, that is, the formation of primitive blood vessels from angioblasts, rather than of angiogenesis, that is, the sprouting of capillaries from preexisting vessels.     

Prevention of endotoxin shock by an antibody against leukocyte integrin {beta}2 through inhibiting production and action of TNF

Septic shock remains a serious disorder associated with highmortality. Accumulating evidence indicates that TNF is a majorand essential mediator of endotoxin shock. We report here thatadministration of an antibody against CD18 dramatically reducedendotoxin-induced shock inrabbits as revealed by preventionof severe hypotension, metabolic acidosis and a pathologicalchange suggestive of disseminated intravascular coagulationwith concomitant inhibition of elevation of plasma TNF activity.The anti-CD18 antibody also inhibited the hypotension inducedby administering recombinant TNF. Furthermore, an antibody againsta ligand for CD18 complexes, intercellular adhesion molecule-1,also prevented TNF-induced shock as well as endotoxin shockinrabbits. These observations suggest that adhesion of leukocytesto endothelium may be of primary importance in the action ofTNF as well as in the production of TNF in vivo and that theantibody against adhesion molecules could be of therapeuticbenefit in life-threatening septic shock in humans.     

Integrins are not involved in the process of human sperm-oolemmal fusion

BACKGROUND: We investigated whether integrins are required forthe human sperm–oocyte binding and fusion processes. METHODS:The expression of several integrin subunits at the human oocyteplasma membrane was investigated using immunofluorescence microscopy,and the functional role of integrins expressed at the humanoocyte surface in sperm–oocyte interaction was studiedusing a zona-free human oocyte binding and fusion assay. A totalof 144 unfertilized oocytes were stained with anti-integrinantibodies and 147 zona-free unfertilized oocytes were inseminatedin the presence of various anti-integrin antibodies that wereexpressed in oocyte plasma membrane. RESULTS: The antibodiesof six integrin subunits (₂, ₃, ₅, ₆, _V, _M) and six integrinsubunits (₁, ₂, ₃, ₄, ₅, ₆) were bound to the surface of fixedunfertilized oocytes. In contrast, the presence of ₁ and ₄ subunitscould not be verified. The human sperm–oocyte bindingwas only partially inhibited by blocking antibodies of ₂, ₃,₅, ₆, _V, _M, ₁, ₂ and ₃ with a maximum of 55% inhibition, butantibodies of ₄, ₅ and ₆ showed no effect on sperm–oolemmalbinding. A similar reduction of the number of fused sperm wasobserved. However, the ratio of fused sperm to total sperm (boundand fused) was not impaired by all integrin antibodies, suggestingthat integrins had no role in the sperm–oolemmal fusionprocess. CONCLUSIONS: These results suggest that one of thebinding mechanisms can be inhibited by integrin antibodies butthat this mechanism does not play an essential role in the humansperm–oolemmal binding and fusion processes. The othermechanisms, insensitive to integrins, may involve both bindingand fusion processes in human oocytes.     

Molecular and biochemical mechanisms ofPasteurella haemolyticaleukotoxin-induced cell death

Pasteurella haemolyticaleukotoxin (LKT) is a member of the RTX family of pore-forming toxins that kill bovine immune cells. Several studies have suggested that RTX toxins kill target cells by the induction of apoptosis. In the present study, BL3 bovine leukaemia cells were exposed to LKT and assessed by molecular and flow cytometric techniques that measure different aspects of apoptotic cell death. The intoxicated cells demonstrated morphological, light scatter and Hoechst 33258 staining characteristics consistent with cells undergoing apoptosis. The cells also exhibited internucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, both indicators of apoptosis. LKT-treated cells bound annexin-V-FITC indicating that phosphatidylserine groups were translocated from the inner to the outer leaflet of the cell membrane. The effect of LKT on cells was dose dependent and inhibitable by incubation with anti-LKT monoclonal antibody. Finally, an early step for induction of apoptosis appears to be the binding of LKT to a β2 integrin since pre-incubating cells with anti-β2 integrin antibodies inhibited LKT-induced apoptosis. This study provides new insights into understanding the pathogenesis of bovine pasteurellosis and could lead to the development of both preventative and therapeutic strategies for disease management.