Hemidesmosome formation is initiated by the beta4 integrin subunit, requires complex formation of beta4 and HD1/plectin, and involves a direct interaction between beta4 and the bullous pemphigoid antigen 180

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the beta4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in beta4 expression. We found that the expression of wild-type beta4 restored the ability of the beta4-deficient cells to form HDs and that distinct domains in the NH2- and COOH-terminal regions of the beta4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the beta4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between beta4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for alpha6 and beta4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of beta4 with BP180. Nevertheless, beta4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of beta4 can interact with the first NH2-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of beta4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of beta4 with other hemidesmosomal components, e.g., BP180, is regulated.     

Abnormal expression of epiligrin and alpha 6 beta 4 integrin in basal cell carcinoma

Lipoprotein lipase (LPL) hydrolyzes the triacylglycerol component of circulating lipoprotein particles, mediating the uptake of fatty acids into adipose tissue and muscle. Insulin is the principal factor responsible for regulating LPL activity in adipose tissue, yet the mechanisms whereby insulin controls LPL expression are unknown. The current studies used wortmannin, a specific inhibitor of phosphatidylinositol (PI) 3-kinase, and rapamycin, a specific inhibitor of activation of phosphoprotein 70 ribosomal protein S6 kinase (p70s6k), to explore some of the components of the insulin signaling pathway controlling LPL activity in adipose cells. Preincubation of isolated rat adipose cells with wortmannin completely abrogated the stimulation of LPL activity by insulin, while preincubation with rapamycin caused approximately a 60% inhibition of insulin-stimulated LPL activity. Thus, the current studies show that the regulation of adipose tissue LPL by insulin is mediated via a wortmannin-sensitive pathway, most likely PI 3-kinase, and that a rapamycin-sensitive pathway, most likely p705s6k, constitutes an important downstream component in the insulin signaling pathway through which LPL is regulated.     

Secondary lymphoid-tissue chemokine (SLC) stimulates integrin alpha 4 beta 7-mediated adhesion of lymphocytes to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) under flow

The attachment of leukocytes to the endothelium is a multistep process that depends upon a very rapid increase in the adhesive activity of leukocyte integrins. A pertussis toxin-sensitive pathway stimulates integrin-dependent lymphocyte adhesion to Peyer's patch high endothelial venules in vivo, but the factors responsible for activating this pathway have not been identified previously. We now report that secondary lymphoid-tissue chemokine (SLC) (also known as 6Ckine, Exodus-2, and thymus-derived chemotactic agent 4), a recently described CC chemokine that is expressed in Peyer's patches and lymph nodes, rapidly activates integrin-mediated lymphocyte adhesion. Immobilized SLC increased the adhesion of HUT-78 T cells and human PBLs to mucosal addressin cell adhesion molecule-1, a protein that is expressed on Peyer's patch and mesenteric lymph node high endothelial venules. This effect of SLC was seen in both static and flow chamber adhesion assays, was mediated by integrin alpha 4 beta 7, and was inhibited by pertussis toxin. The other CC chemokines tested did not increase adhesion to mucosal addressin cell adhesion molecule-1. SLC had a greater effect on naive CD4+ T cells than on memory CD4+ T cells; CD8+ T cells, B cells, and NK cells were also responsive to SLC. SLC is likely to play an important role in regulating the recruitment of lymphocytes to Peyer's patches and lymph nodes.     

The integrin alpha6beta4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures

Functional studies on the alpha6beta4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the alpha6beta4 integrin in clone A cells, a colon carcinoma cell line that expresses alpha6beta4 but no alpha6beta1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the alpha6beta4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the alpha6beta4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although beta1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the alpha6beta4 integrin and F-actin was seen. An association between alpha6beta4 and F-actin is supported by the fact that alpha6beta4 integrin and actin were released from clone A cells by treatment with the F-actin- severing protein gelsolin and that alpha6beta4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, alpha6beta4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited alpha6beta4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an alpha6-specific antibody. Together, these results indicate that the alpha6beta4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures.     

Activation status and function of the VLA-4 (alpha4beta1) integrin expressed on human melanoma cell lines

A highly sensitive microspectrophotometer was developed to measure spectral changes of oxyhemoglobin (oxy Hb) in single red blood cells (RBC) incubated with stimulated macrophages as a model of nitric oxide (NO) dependent cytotoxicity. Our microspectrophotometer, using a modified acousto-optic tunable filter (AOTF) and a 2-dimensional CCD array, allows fast spectrophotometric data acquisition. Human RBC treated with various concentrations of NO showed spectral changes due to the conversion of oxy Hb to methemoglobin (met Hb), in which the change in absorption differences at alpha (557 590 nm) and beta (542-525 nm) bands showed a linear relationship with the concentration of NO up to 100 microM. In contrast to highly diffusible NO, nitrite ions (NO2-) seem to enter RBC very slowly, resulting in negligible formation of met Hb in the presence of 5 mM glucose even during a prolonged incubation period. RBC were incubated with murine macrophages with and without lipopolysaccharide (LPS) in the presence of glucose for 24 and 40 h and subjected to the microspectrophotometric assay. The RBC incubated with LPS-stimulated macrophages showed significant changes in the spectrum due to NO-dependent conversion of oxy Hb to met Hb, which corresponded to the spectral changes of RBC treated with a several times higher concentration of NO than that in the culture medium. The trapping efficiency was calculated from the amounts of the NO released from macrophages and of the met Hb formed in the RBC, which gave a high efficiency (43%). The results suggest that RBC trap NO directly by cell cell interaction with macrophages. This spectrophotometric system is available for use with just a few drops of samples to study NO-specific cytotoxicity as a model of RBC without the use of any chemical reagent, in parallel with microscopic observations on changes of the cellular morphology under physiological conditions, such as membrane damage leading to hemolysis, adherence, and phagocytosis.     

Adhesion to laminin is down-regulated upon retinoic acid-induced F9 cell differentiation: a role for alpha 6/beta 1 integrin

F9 mouse teratocarcinoma cells have a high capacity to adhere to laminin and we identified alpha 6/beta 1 integrin as the principal laminin-binding protein present in these cells. F9 cells differentiated into parietal endoderm when monolayer cultures were treated with retinoic acid and dibutyryl cyclic AMP. In this process a decreased adherence to laminin was observed due to a lower expression of alpha 6/beta 1 integrin on the cell surface.     

Increased expression of intercellular adhesion molecule 1, CD11/CD18 cell surface adhesion glycoproteins and alpha 4 beta 1 integrin in a rat model of chronic interstitial lung fibrosis

The expression of the intercellular adhesion molecule 1 (ICAM-1), and the integrins CD49, CD11b/c, and CD11a (LFA-1 alpha chain) was analyzed in an experimental model of pulmonary fibrosis. Adult rats were exposed to 75% oxygen during 10 weeks, and to 2.0 mg/kg of paraquat twice weekly. Rats were sacrificed at 2 days, and at 2 and 10 weeks after the first injection of paraquat. Lungs were fixed in 4% paraformaldehyde and used for histology and immunohistochemistry. At 2 days the lungs showed a diffuse inflammation composed of a mixed polymorphonuclear and mononuclear cell infiltrate. Afterwards, the inflammatory process was predominantly mononuclear, and an increasing fibroblast proliferation was observed. Early inflammatory events (48 h) correlated with a moderate increased expression of ICAM-1, LFA, and CD11b/c in epithelial cells as well as a pronounced expression of ICAM-1 and CD11b/c in macrophages. At 2 and 10 weeks, there was a progressive increased expression of CD11b/c and ICAM-1 by macrophages, as well as of LFA in epithelial cells, and of ICAM-1 and CD49 by epithelial and interstitial cells. Lymphocytes showed a slight increased expression of LFA at 2 weeks, and of CD49 at 2 and 10 weeks. These results suggest that macrophages expressing ICAM-1, CD11b/c, and CD49 are involved in the earlier and late phases of the disease whereas fibroblast and epithelial cells expressing ICAM-1 and CD49 might play a role in the cell interactions involved in the fibrotic phase.     

Novel ITGB4 mutations in a patient with junctional epidermolysis bullosa-pyloric atresia syndrome and altered basement membrane zone immunofluorescence for the alpha6beta4 integrin

Immunofluorescence studies of junctional epidermolysis bullosa with pyloric atresia (JEB-PA) have suggested abnormalities in the expression of the alpha6 beta4 integrin, an integral component of hemidesmosomes. In this study, we examined a family with two affected individuals with JEB-PA for mutations in the ITGA6 and ITGB4 genes which encode the alpha6 and beta4 integrin polypeptides, respectively. Mutation detection strategy based on PCR amplification of genomic DNA, followed by heteroduplex analysis and direct nucleotide sequencing, did not reveal sequence variants in ITGA6. Putative pathogenic mutations, however, were identified in both ITGB4 alleles. Specifically, the proband was a compound heterozygote for a 1-bp maternal deletion, 3434delT, and an 8-bp paternal deletion, 4050de18. Both mutations result in a frameshift and premature termination codon downstream from the deletion. At the protein level, immunofluorescence of the skin of the proband revealed negative staining for the integrin alpha6 and markedly reduced staining for the beta4 subunit. Thus, the results support the notion of close association of the alpha6 beta4 integrin subunits and further attest to the critical role of this integrin in providing physiologic stability to the dermal-epidermal junction.     

Roles of hydrogen bonding residues in the interaction between the alpha and beta subunits in the tryptophan synthase complex. Asn-104 of the alpha subunit is especially important

The interaction of the alpha subunit with the beta2 subunit of tryptophan synthase is known to be necessary for the activation of each subunit and for the catalytic efficiency of the alpha2beta2 complex. To elucidate the roles of hydrogen bonds in the interaction site between the alpha and beta subunits for subunit association, eight mutant alpha subunits at five hydrogen bonding residues (N104D, N104A, N108D, N108A, E134A, E135A, N157D, and N157A) were constructed, and the thermodynamic parameters of association with the beta subunit were obtained using a titration calorimeter. The N104D and N104A mutations remarkably decreased the stimulation activities, the association constants, and the association enthalpies. Although the association constant and the stimulation activities of E134A were reduced in the absence of salt, the change in the association enthalpy was relatively small, and the addition of salt could repair its defects. The substitutions at positions 135 and 157 did not affect the stimulation activity and decreased the Gibbs energy of association corresponding to the defect in 1 mol of hydrogen bond. The present results suggest that the alpha subunit which has a mutation at position 104 cannot fold into an intact conformation upon complex formation, resulting in reduced stimulation activities. The hydrogen bond with Asn-104, which is a conserved residue among 16 microorganisms, was especially important for alpha/beta interaction and mutual activation.     

Rat neutrophils express alpha4 and beta1 integrins and bind to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1)

The alpha4 integrins, which are constitutively expressed on all human leukocyte subtypes except neutrophils, interact with vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule (MAdCAM-1) on endothelium to mediate selective recruitment of leukocyte subpopulations, other than neutrophils, to sites of inflammation. However, here we report that a different paradigm of leukocyte recruitment may exist in the rat. Flow cytometric analysis of rat neutrophils using a panel of monoclonal antibodies which recognize rat alpha4 and beta1 integrins showed consistent, low levels of expression. Although alpha4 was expressed at lower levels on neutrophils than all other rat leukocytes, this level of expression was sufficient to mediate significant levels of alpha4- and beta1-dependent neutrophil adhesion to rat and human VCAM-1, and alpha4-dependent, but beta1-independent, adhesion to human MAdCAM-1. These data suggest that rat neutrophils, unlike other species, may use alpha4 integrins to traffic to sites of inflammation in vivo.     

Platelet/polymorphonuclear leukocyte interaction in dynamic conditions: evidence of adhesion cascade and cross talk between P-selectin and the beta 2 integrin CD11b/CD18

Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence-activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins "activation reporter." When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP- or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.     

Differential effects of the integrins alpha9beta1, alphavbeta3, and alphavbeta6 on cell proliferative responses to tenascin. Roles of the beta subunit extracellular and cytoplasmic domains

Members of the integrin family manifest considerable overlap in ligand specificity, and many cells have the capacity to express multiple integrin receptors for the same ligand. For example, at least 5 different integrins recognize tenascin as a ligand, and 4 of these bind to the same region of the protein, the third fibronectin type III repeat (TNfn3). We utilized colon carcinoma cells (SW480) that do not normally attach to TNfn3 to examine the possibility that ligation of different integrin receptors for this ligand would induce different effects on cell behavior and intracellular signaling. Heterologous expression of the tenascin receptors alphavbeta3 and alpha9beta1 produced comparable effects on cell adhesion and spreading on TNfn3, but alphavbeta3-transfectants proliferated considerably better on each concentration examined. alphavbeta6-transfectants attached (although less avidly), but completely failed to spread or proliferate. Expression of a chimeric beta subunit composed of the beta3 extracellular domain fused to the beta6 transmembrane and cytoplasmic domains resulted in adhesion and spreading similar to that seen with beta3-transfectants, but considerably less proliferation. When the same cell lines were plated on fibronectin, alphavbeta6-transfectants spread and proliferated as well as cells transfected with the chimeric beta3/beta6 subunit, but, again, neither cell line proliferated as well as cells expressing alphavbeta3. Cell proliferation was always associated with spreading and with phosphorylation of the focal adhesion kinase, paxillin, and the mitogen-activated kinase, Erk2, but cell attachment in the absence of spreading or proliferation was not associated with phosphorylation of any of these proteins. These data suggest that different integrin receptors for a single ligand can produce markedly different effects on cell proliferation, and that both the extracellular and cytoplasmic domains of integrin beta subunits contribute to these differences.     

Differential effects of antibodies to vascular cell adhesion molecule-1 and distinct epitopes of the alpha4 integrin in HgCl2-induced nephritis in Brown Norway rats

Four distinct epitopes (A, B1, B2, and C) have been functionally defined on the human alpha4 integrin. In this study, two cross-reactive antihuman alpha4 monoclonal antibodies (mAb) (HP2/1 and HP2/4 specific for epitopes B1 and B2, respectively) were used to functionally characterize the rat VLA-4 subunit and to define similar functional epitopes in this rodent species. It was found that B1 and B2 anti-alpha4 mAb completely block adhesion to fibronectin, but the inhibition of adhesion to vascular cell adhesion molecule-1 (VCAM-1) with HP2/1 mAb was lower than with HP2/4 mAb. It was also observed that epitope B2 HP2/4 mAb induced homotypic aggregation in rat lymphocytes, whereas epitope B1 HP2/1 mAb did not. Using the HgCl2 model of nephritis, this study shows the protective effect of both anti-alpha4 mAb against infiltration of the renal interstitium by leukocytes. Nevertheless, HP2/1 mAb, but not HP2/4 mAb, virtually abolished the anti-glomerular basement membrane antibody synthesis and glomerular deposits. These findings indicate the dual but independent role played by alpha4 integrins in both extravasation of leukocytes and in the production of antibodies. Finally, this study demonstrates that anti-rat VCAM-1 mAb showed a positive reactivity of the renal vascular endothelium and, most importantly, that administration of anti-VCAM-1 antibodies completely abrogated the interstitial cell infiltrates without affecting anti-glomerular basement membrane antibody production. These results confirm the important role played by VLA-4/VCAM-1 pathway in leukocyte infiltration, and further support the dual and independent role of alpha4 integrins in both renal infiltration and autoantibody synthesis in this model of renal disease.     

Neural crest cell interactions with laminin: structural requirements and localization of the binding site for alpha 1 beta 1 integrin

We have identified the sites of neural crest cell interaction with laminin in vitro by examining their ability to attach to and migrate on proteolytic fragments of the molecule and the ability of fragment-specific antibodies to inhibit these interactions. The binding site on laminin was localized to the E8 domain on the long arm of laminin, as well as the T8' fragment within this domain, but not the E1', E3, or E4 fragments. Only subfragments containing the carboxy-terminal rod-like portion of the A chain plus the corresponding B1 and B2 chains retained the attachment-promoting activity of the parent E8 fragment. In addition, interactions required maintenance of the triple-stranded and alpha-helical coiled-coil structure of this domain. Reduction and alkylation of laminin and the E8 and T8 fragments significantly reduced neural crest cell attachment and migration. An antiserum against chick alpha 1 integrin reduced migration and adhesion of neural crest cells on an intact laminin-nidogen complex, the E8 fragment, and all its active subfragments. Furthermore, we observed that neural crest cells modified laminin substrata prepared in the absence of divalent cations. Early stable attachment to these substrata was mediated by an integrin other than alpha 1, whereas later attachment and migration were mediated by alpha 1 integrins. Our results suggest that neural crest cells selectively bind to the B1-A-B2 mid-portion (T8') of the E8 domain of laminin, requiring structural integrity of this region and that they modify laminin substrata as a result of prolonged cell-matrix interactions.     

Ser752 mutation to Pro or Ala in the beta3 integrin subunit differentially affects the kinetics of cell spreading to von Willebrand factor and fibrinogen

The beta3 cytoplasmic domain of the alpha v beta3 integrin is essential for intracellular signals required for cytoskeletal rearrangements. Expression of beta3Ser752Pro mutation in heterologous cells profoundly affects cell spreading and beta3 localization into focal contacts. However, the beta3Ser752Ala substitution mostly restores normal integrin functions, suggesting that the presence of Pro is responsible for the receptor's loss of function. To further assess the role of the Ser752 of the beta3 cytoplasmic domain in the cytoskeletal organization of adherent cells, we developed a computer-assisted method of image analysis allowing the automatic classification of spread cells according to the quantitative analysis of their cell morphology. We compared adhesion and spreading to von Willebrand factor (vWF) or fibrinogen (Fg) of cells expressing beta3 wild type, beta3Ser752Pro or beta3Ser752Ala mutated integrin subunit as a chimeric alpha v beta3 receptor. The beta3Ser752Ala substitution did not impair the general ability of cells to spread, but resulted in a delayed and reduced spreading on both vWF and Fg. Moreover, the beta3Ser752Ala mutation produced modifications of the morphology of spread cells, suggesting a disorganization of their cytoskeleton. Attachment studies showed that the beta3Ser752Ala mutation did not modify the capacity of cells to attach to the substrate, indicating no change in the ligand binding affinity of the alpha v beta3 integrin. Furthermore, we identified a slight defect of beta3Ser752Pro cell attachment to vWF and Fg, beside their impairment of spreading. Taken together, these results suggest a role of Ser752 of the beta3 cytoplasmic domain in the optimal cytoskeletal organization of adherent cells.     

Exposure to prenatal nicotine transiently increases neuronal nicotinic receptor subunit alpha7, alpha4 and beta2 messenger RNAs in the postnatal rat brain

This study determined the effects of prenatal nicotine exposure (2 mg/kg/day) in Sprague Dawley CD rats via subcutaneously implanted osmotic minipumps, during gestational days 7-21, on postnatal levels of neuronal nicotinic receptor alpha4, alpha7 and beta2 subunit messenger RNAs. Northern analysis of postnatal day 1, 7, 14 and 28 hippocampal/septal and cortical total RNA using alpha-[32P]dCTP-labeled alpha4, alpha7 and beta2 complementary DNA probes identified a single (5.7-kb) alpha7 messenger RNA, three (2.4-, 3.8- and 8.0-kb) alpha4 messenger RNAs and four (3.7-, 5.0-, 7.5- and 10.0-kb) beta2 messenger RNAs. In comparison to prenatal saline, prenatal nicotine produced several significantly higher messenger RNA levels (cortical: 5.7-kb alpha7, 2.4-, 3.8- and 8.0-kb alpha4, 10.0-kb beta2; hippocampal/septal: 2.4- and 8.0-kb alpha4); these increases occurred predominantly on, but were not restricted to, postnatal day 14. Effects of nicotine were generally resolved by postnatal day 28. Collapsing the data across sex and age, a significant treatment effect indicated that hippocampal/septal and cortical 8.0-kb alpha4 messenger RNA levels and 10.0-kb beta2 messenger RNA levels were significantly higher following prenatal nicotine exposure. This is the first study indicating that prenatal nicotine produces alterations in developing postnatal rat neuronal nicotinic receptor messenger RNA levels, possibly by premature stimulation of neuronal nicotinic receptors. These results further implicate the teratogenic potential of nicotine in postnatal neuronal development.     

Expression of cell adhesion molecules at the surface of in vitro human immunodeficiency virus type 1-infected human monocytes: relationships with tumor necrosis factor alpha, interleukin 1beta, and interleukin 6 syntheses

Further evidence suggests that cell adhesion molecules (CAMs) expressed on the surface of human immunodeficiency virus type 1 (HIV-1)-infected cells are regulated during lentiviral infection. To address this hypothesis we have investigated the kinetic pattern of CAM expression at the surface of HIV-1Ba.L-infected human monocytes during the first 72 hr of infection. A significantly lower expression of CD18 and CD54 as well as a decrease in CD44 expression level were observed at the surface of infected monocytes when compared with mock-infected cultures. No modification of CD11a, CD11b, CD11c, CD58, and CD62L expression was detected. Except for CD18, the expression of which at the cell surface is decreased, no modification of CD44 and CD54 expression was observed after heat-inactivated HIV-1 treatment of monocytes. Investigation of soluble forms of CAMs (sCAMs) and cytokine production in the culture supernatants of infected monocytes showed a peak of sCD44, TNF-alpha, IL-1beta, and IL-6 release between 2 and 24 hr after infection. Treatment of monocytes with monoclonal antibodies (MAbs) against CAMs showed that engagement of some CAMs may trigger TNF-alpha and IL-1beta production. In addition, pretreatment of infected monocytes with a TNF-alpha synthesis inhibitor, RP 55778, or with MAbs directed against IL-1beta, confirmed the role of TNF-alpha and IL-1beta in the regulation of CD18, CD44, and CD54 expression.     

Upmodulation of alpha v beta 1 integrin expression on human tumor cells by human interleukin for DA cells/leukemia inhibitory factor and oncostatin M: correlation with increased cell adhesion on fibronectin

Integrins belong to a large family of heterodimeric membrane glycoproteins which mediate cell-cell or cell-extracellular matrix interactions. These interactions could play a major role during the migration of tumor cells across the extracellular matrix and vascular endothelium and would thus appear to be requisite for the metastatic process. Pretreatment of the Foss human melanoma cell line with HILDA/LIF or OSM, two cytokines involved in acute-phase response, increased the expression of membrane alpha v beta 1 1.5-2-fold. The same phenomenon was observed on the SK-N-SH human neuroblastoma cell line. alpha v beta 1 upmodulation was concomitant with improved tumor cells attachment to the fibronectin matrix. This greater adhesion of tumor cells to fibronectin was inhibited by specific monoclonal antibodies against alpha v or beta 1 integrin subunits. Similar results were obtained after TNF-alpha treatment. Our findings demonstrate the ability of HILDA/LIF and OSM to modulate tumor cell capacity to adhere to the matrix component, suggesting a potential role for these cytokines in modulation of tumoral progression.