Highly stoichiometric, stable, and specific association of integrin alpha3beta1 with CD151 provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration.

Here we describe an association between alpha3beta1 integrin and transmembrane-4 superfamily (TM4SF) protein CD151. This association is maintained in relatively stringent detergents and thus is remarkably stable in comparison with previously reported integrin-TM4SF protein associations. Also, the association is highly specific (i.e., observed in vitro in absence of any other cell surface proteins), and highly stoichiometric (nearly 90% of alpha3beta1 associated with CD151). In addition, alpha3beta1 and CD151 appeared in parallel on many cell lines and showed nearly identical skin staining patterns. Compared with other integrins, alpha3beta1 exhibited a considerably higher level of associated phosphatidylinositol-4-kinase (PtdIns 4-kinase) activity, most of which was removed upon immunodepletion of CD151. Specificity for CD151 and PtdIns 4-kinase association resided in the extracellular domain of alpha3beta1, thus establishing a novel paradigm for the specific recruitment of an intracellular signaling molecule. Finally, antibodies to either CD151 or alpha3beta1 caused a approximately 88-92% reduction in neutrophil motility in response to f-Met-Leu-Phe on fibronectin, suggesting an functionally important role of these complexes in cell migration.

Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or -independent mechanisms.

Work reported here investigated aspects of macrophage-mediated tumor cell death, in particular the role of apoptosis as a mechanism for nitric oxide (NO)-mediated macrophage tumor cytotoxicity. Nitric oxide induced apoptosis in P815 cells in macrophage P815 cocultures where fragmentation of tumor cell [3H]thymidine-labeled DNA preceded cell lysis (as measured by 51Cr release), paralleled nitrite accumulation, and was prevented by a specific inhibitor of NO synthase, N-MMA. DNA from P815 cells separated from macrophages in culture by a cell-impermeable membrane or exposed to authentic NO gas showed the pattern of internucleosomal cleavage that is characteristic of apoptosis. Additionally, culture of P815 cells with the NO donor sodium nitroprusside was followed by DNA fragmentation. Macrophages also induced apoptosis in L929 cells but, in this case, apoptosis was NO independent and partially inhibited in cocultures by an antitumor necrosis factor alpha monoclonal antibody. The anti-tumor necrosis factor alpha monoclonal antibody fully prevented apoptosis when macrophages and L929 were separated by a cell-impermeable membrane. Exposure of L929 cells to NO gas or sodium nitroprusside did not result in their apoptotic death. Like other immune cytotoxic cells, macrophages can determine tumor cell death through the induction of apoptosis and do so through more than one effector mechanism.

Macrophage phagocytosis of wound neutrophils.

Resolution of acute inflammation is thought to require the recognition and phagocytosis of apoptotic neutrophils (PMN) through receptor-ligand interactions with macrophages (Mphi). This hypothesis was tested in rat wounds by quantifying apoptosis in freshly harvested and aged-in-culture PMN taken from wounds 1-3 days after injury and by using these wound PMN as phagocytic targets for wound, immune-activated peritoneal, and resident peritoneal Mphi. Less than 6% of freshly harvested PMN exhibited characteristics of apoptosis. On aging in culture, day 1 PMN did not undergo apoptosis, whereas 41+/-1 and 29+/-1% of day 2 and 3 PMN, respectively, developed apoptosis, which corresponded to increased ingestion by Mphi. All three Mphi populations engaged different receptor-ligand pairs for the recognition and phagocytosis of PMN. Results indicate the resistance of early wound PMN to age-induced apoptosis, demonstrate wound-Mphi phagocytosis of wound PMN, and identify distinct receptor utilization by wound and other Mphi to ingest wound PMN.

NO is not sufficient to explain maximal cytotoxicity of tumoricidal macrophages against an NO-sensitive cell line.

Nitric oxide (NO) is a macrophage cytotoxic effector. Results presented here, however, demonstrate that NO does not fully explain macrophage cytotoxicity against NO-sensitive cells because (1) inhibition of NO production by activated macrophages reduces, not eliminates, cytotoxicity; (2) NO produced chemically in amounts equimolar to those released from macrophages fails to lyse P815 cells; and (3) macrophages isolated from wounds 10 days after injury generate NO just as tumoricidal activated macrophages but are not tumor cytotoxic. The noncytotoxic nature of these wound-derived macrophages is not explained by the release of inactive forms of NO, because they suppress lymphocyte proliferation in an NO-dependent manner, nor by the production of cytoprotective molecules, because their addition to activated macrophage-tumor cell cocultures does not quench cytotoxicity. Interestingly, cytotoxicity can be aroused in day 10 wound-derived macrophages by culture with lipopolysaccharide, and macrophages harvested earlier in the development of the wound are cytotoxic. By generating NO but not killing an NO-sensitive cell, day 10 wound-derived macrophages demonstrate that NO production is not sufficient to account for the killing of an NO-sensitive tumor by macrophages.

Increased neutrophil motility by beta-glucan in the absence of chemoattractant.

Systemic candidasis is a life-threatening complication of antibiotic and immunosuppressive therapies and can alter host defense mechanisms through pathways that are poorly understood. Promotion of polymorphonuclear leukocyte (PMN) chemotaxis by beta-glucan towards fMLP or IL-8 gradients demonstrates a fundamental effect on host defenses by pathogenic fungi. The aim of the present study was to determine whether recognition of beta-glucan is sufficient to alter PMN motility in the absence of agonists of G-coupled protein chemotactic receptors. Present findings demonstrate a profound increase in PMN motility by beta-glucan supplementation of a fibronectin substratum in an underagarose migration assay. Motility on beta-glucan included a 3-fold increase in distance of migration, as well as a 5-fold increase in the number of PMNs recruited into the motile phase as compared to motility on fibronectin alone. This promotion of motility is determined by the beta2 integrin complement receptor 3 (CR3) (CD11b/CD18) rather than the beta1 integrin very late antigen 3 (VLA-3), which mediates chemotaxis on beta-glucan-supplemented matrix towards fMLP. PMN motility on beta-glucan-supplemented fibronectin was selectively decreased by inhibitors of pp60 src and ras, whereas motility was promoted by inhibition of p38-MAPK. No effect of these inhibitors was seen on PMNs migrating on fibronectin alone. Migration on beta-glucan-supplemented fibronectin, but not on fibronectin alone, was negatively regulated by protein kinase C (PKC) or cAMP activation. These findings indicate that beta-glucan is sufficient to alter the migratory capacity of PMN in the absence of costimulation by fMLP. Enhanced PMN migration on beta-glucan is mediated through specific integrins and second messenger pathways that are distinct from those utilized by PMNs migrating in the absence of beta-glucan.

Wound-induced tumor progression: a probable role in recurrence after tumor resection.

OBJECTIVE: To determine the effect of several wound factors on melanoma growth in a mouse model. DESIGN: Cohort analytic study. SETTING: Animal research facility of Roger Williams Medical Center, Providence, RI. STUDY GROUP: Seventeen groups of 5 C57BL/6 mice each. INTERVENTIONS: A surgical wound was created in 1 hind limb, after which different concentrations of B16F10 melanoma cells were injected in adjacent subcutaneous tissue. The nonwounded hind limb in the same mouse served as a control. In this fashion, a critical tumor cell dose was determined that showed tumor growth in the wounded but not the control hind limb. Tumor growth in control hind limbs then was compared with that in the "artificially wounded" hind limbs, which were co-injected with mouse wound fluid or growth factors. Early (day 1) and late (day 10) wound fluids and tumor growth factor beta (TGF-beta), basic fibroblast growth factor (bFGF), both combined, and interleukin 6 (IL-6) were used. MAIN OUTCOME MEASURE: Wound factors increase tumor growth, indicating potentiation of tumor recurrence at a surgical wound. RESULTS: The critical tumor cell dose was 10(3) cells. All growth factors and both wound fluids showed increased tumor growth over time except IL-6. Hind limbs injected with early wound fluid showed increased tumor growth over time when compared with those injected with late wound fluid (P<.001), TGF-beta (P<.001), bFGF (P<.001), and IL-6 (P<.001). Combined TGF-beta and bFGF co-injection resulted in increased tumor growth compared with TGF-beta (P<.001) and bFGF (P<.001), but did not differ significantly from early wound fluid (P<.07). CONCLUSIONS: The healing wound and its mediators in wound fluid or purified growth factors significantly enhanced tumor growth. Combining TGF-beta and bFGF increased tumor growth to a level closer to wound fluid. The inflammatory response provoked by wound healing mediators may be an important mechanism in tumor growth after ablative surgery.

Interleukin-6 production by rat hepatocellular carcinoma cells is associated with metastatic potential but not with tumorigenicity.

BACKGROUND: The phenotypic characteristics that allow some tumor cells to metastasize have not been fully identified. The production and/or response of tumor cells to various growth factors have been shown to distinguish cells of differing metastatic potentials. OBJECTIVES: To determine (1) whether rat hepatocellular carcinoma cell lines produce interleukin-6 (IL-6) and (2) whether production of IL-6 correlates with either metastatic potential or tumorigenicity. METHODS: The clonal cell lines 1682.C.2.9.L0 (poorly metastatic) and 1682.C.2.9.L10 (highly metastatic) were selected from a parental hepatocellular carcinoma induced in ACI rats by feeding an ethionine-containing diet and adapted to growth in vitro. RESULTS: Both cell lines resulted in primary tumors with equal frequency and developed a 40-mm nodule in a similar period time, when an inoculum of 5 X 10(6) cells was injected subcutaneously; however, only L10 cells metastasized to the lung. These cell lines did not demonstrate differential expression of several antigens noted to correlate with metastatic potential, including CD44 variant glycoprotein, p53, transferrin receptor, and E-cadherin. In contrast, L0 cells produced less than 10 U of IL-6 per milliliter in culture (as determined by bioassay using 7TD1 cells), whereas L10 cells released more than 95 U of this cytokine per milliliter under identical culture conditions (P<.01, Student's t test). In addition, serum concentrations of IL-6 were elevated in animals bearing L10-induced primary tumors but not in those with L0-induced tumors of comparable mass. Exogenous addition of IL-6 to both tumor cell lines had no effect on the rate of growth in vitro, supporting the similar the tumorigenic potentials observed in vivo. CONCLUSION: Excess IL-6 production appears to identify cells with metastatic potential and does not appear to be essential to the establishment of a primary tumor.

The effect of surgical wounding on tumour development.

For more than a century, a role for wound healing in the outgrowth of tumours has been implied based on observations in both experimental and clinical studies. Wound healing can be divided into stages of inflammatory, proliferative, repair and remodelling processes. Through proper regulation of activation of epithelial, endothelial and inflammatory cells, platelets and fibroblasts, and the production of growth factors, wounds heal and the various cell types resume their normal function. In tumour growth, similar processes of cell activation and growth factor production are observed. These processes are, however, differently regulated leading to ongoing cellular activation. In recent years, growth factors such as EGF, TGF-alpha and TGF-beta, bFGF, IGF I and II, and PDGF have been identified to play a role in the different stages of wound healing. In addition, some of these factors have now been identified as also being involved in the outgrowth of tumours. In this review, cell types involved in wound healing and tumour growth, as well as the growth factors and cytokines they produce and the role of the extracellular matrix, extensively present in both conditions, are being discussed. A better understanding of the time interval during which the sequelae of events in wound healing occur in relation to the time interval of tumour recurrence may be the basis for defining new therapeutic strategies that can interfere with tumour outgrowth without affecting wound healing processes. These new therapeutic approaches may be of importance especially after surgery or other invasive (diagnostic) procedures in cancer patients.

The search for H-2 complementation affecting the anti-Thy-1 response in mice: a progress report.

It has been postulated that combinatorial Ia molecules of IAb/Iad heterozygotes are involved in the recognition of the Thy-1 alloantigens. These molecules could be considered the products of the complementary Ir-Thy-1 genes. In present studies, no complementation was found among 37 different H-2 heterozygous F1 hybrids except those carrying IAb/IAd haplotypes. However, in two instances involving hybrids carrying IAr alleles anti-Thy-1 responses were found to be better than expected. The possible mechanism(s) of such responses is currently under investigation.

Role of nitric oxide in mediation of macrophage cytotoxicity and apoptosis.

Macrophages can recognize and eliminate tumor cells. To this effect, these cells use a variety of cytotoxic effectors. Recent work has paid particular attention to nitric oxide (NO) and its metabolic by-products in mediating macrophage tumor cytotoxicity. Moreover, work from this and other laboratories have indicated that macrophage-dependent, NO mediated tumor cell death meets the morphologic and molecular criteria that define apoptotic cell death. This review will initially discuss the characteristics of macrophage tumor cytotoxicity and the potential mechanisms by which NO can induce apoptosis in tumor cells. In addition, observations of spontaneous and acquired resistance to NO will be analyzed. Lastly, the relevance of results obtained using animal cells to the biology of the human macrophage will be considered.

Nitric oxide in inflammation and immunity.

Few discoveries have had as comprehensive an impact on the understanding of cellular physiology as the production of nitric oxide (NO.) from the terminal guanido amino group of L-arginine through nitric oxide synthases. The sheer volume of data presently coming forth on the physiology and pathophysiology of NO. ensures that any attempt at a comprehensive review will result in a simple snapshot of the event, soon to be outdated.

Intracellular trafficking of cell surface sialoglycoconjugates.

Recent reports have suggested that the majority of the molecular traffic through the Golgi apparatus is comprised of recycling, rather than newly synthesized, molecules. To evaluate the importance of this recycling pathway in greater detail, we examined the internalization and recycling of cell surface glycoproteins on EL-4 cells, a murine T-cell lymphoma, using sialic acids as covalent markers. Sialic acids were removed from the surface of living cells by exhaustive treatment with Vibrio cholerae sialidase at 4 degrees C and shown to be derived primarily from glycoproteins (93%), with only a small amount from glycolipids (7%). Cells were recultured at 37 degrees C over time and monitored for the resialylation of the cell surface using a sensitive high pressure liquid chromatography adaptation of the thiobarbituric acid assay for sialic acids. The return of sialic acid to the cell surface was found to be contingent upon de novo protein synthesis indicating that the bulk of plasma membrane sialoglycoconjugates do not recycle to an endogenous sialyltransferase-containing compartment for oligosaccharide reprocessing. Identical results were found for K562 cells, a human erythroleukemia cell line. The movement of specific glycoproteins was followed using the enzyme rat liver alpha 2-6Gal beta 1-4GlcNAc sialyltransferase together with CMP-[3H]NeuAc as an impermeant probe of the cell surface. Surface sialoglycoproteins were internalized slowly, a process unaffected by cycloheximide treatment. Only a few of these internalized glycoproteins were found to return to a trans-Golgi compartment followed by recycling to the cell surface. Taken together, these data indicate that the majority of replacement of sialic acids on the cell surface is due to de novo synthesis of glycoproteins and that only a small number of glycoproteins recycle through a trans-Golgi compartment.

New Thy-1- and H-2-congenic strains of mice and their application in studies on the mechanism of anti-Thy-1.1 response.

The magnitude of the primary humoral anti-Thy-1.1 responses was measured by the number of PFC in spleens of B10.S(7R) mice and some of their H-2 heterozygous F1 hybrids immunized with thymocytes of either AKR.TH or B10.S(7R)-Thy-1a strain. These two of the five newly developed strains are non-H-2 incompatible and non-H-2 compatible with B10.S(7R) mice, respectively. The low anti-Thy-1.1 responses of B10.S(7R) mice and their F1 hybrids after immunization with thymocytes from non-H-2 compatible B10.S(7R)-Thy-1a donors indicated that the I-As "allele" of B10.S(7R) mice and the I-A "alleles" of other parental strains tested did not produce a complementary effect that is required for non-H-2 compatible thymocytes to elicit a good anti-Thy-1 response.

Recycling cell surface glycoproteins undergo limited oligosaccharide reprocessing in LEC1 mutant Chinese hamster ovary cells.

The ability of particular cell surface glycoproteins to recycle and become exposed to individual Golgi enzymes has been demonstrated. This study was designed to determine whether endocytic trafficking includes significant reentry into the overall oligosaccharide processing pathway. The Lec1 mutant of Chinese hamster ovary (CHO) cells lack N -acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface expression of incompletely processed Man5GlcNAc2 N -linked oligosaccharides. An oligosaccharide tracer was created by exoglycosylation of cell surface glycoproteins with purified porcine GlcNAc-TI and UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the next enzyme in the Golgi processing pathway of complex N -linked oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface glycoproteins were included in this endocytic pathway indicates a common intracellular compartment into which endocytosed cell surface glycoproteins return. Significantly, no evidence was found for continued oligosaccharide processing consistent with transit through the latter cisternae of the Golgi apparatus. These data indicate that, although recycling plasma membrane glycoproteins can be reexposed to individual Golgi-derived enzymes, significant reentry into the overall contiguous processing pathway is not evident.