Tumor necrosis factor-alpha provokes a hypertrophic growth response in adult cardiac myocytes.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine with a broad range of concentration-dependent effects. The recent observation that TNF-alpha is expressed by the cardiac myocyte after certain forms of stress suggests that TNF-alpha might contribute to the maintenance of normal tissue homeostasis after environmental injury. Accordingly, the purpose of this study was to examine the effects of TNF-alpha on protein synthesis in cultured adult cardiac myocytes. METHODS AND RESULTS: Cultured adult feline cardiac myocytes were stimulated with 10 to 1000 U/mL TNF-alpha to examine the effects of this cytokine on the rate of protein synthesis and degradation. Stimulation with TNF-alpha led to an accelerated rate of general protein synthesis and a time-dependent decrease in protein degradation in adult cardiac myocytes. The specificity of these findings was demonstrated by studies in which the effects of TNF-alpha on protein synthesis were blocked by a neutralizing anti-TNF-alpha antibody as well as studies in which TNF-alpha-conditioned medium had no effect on protein synthesis in myocytes. In addition to the TNF-alpha-induced increase in the general protein synthesis, stimulation with TNF-alpha led to a 2.4-fold increase in net actin protein synthesis and a 3.3-fold increase in net myosin heavy chain synthesis. Finally, the effects of TNF-alpha on adult cardiac myocytes were shown to be dependent on cell-substrate interaction, suggesting that the cell signaling pathways used by TNF-alpha are dependent on a preserved interaction between cell integrins and the extracellular matrix. CONCLUSIONS: The observation that TNF-alpha provokes a hypertrophic growth response in cardiac myocytes suggests that TNF-alpha may play an important role in myocardial homeostasis after environmental stress.

Liposomal muramyl tripeptide upregulates adhesion molecules on the surface of human monocytes.

We previously demonstrated that liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE), a biologic response modifier now undergoing phase III clinical trial in osteosarcoma, upregulated monocyte expression of several cytokines' mRNA and the subsequent production of these proteins. In the present work, we investigated whether L-MTP-PE upregulated adhesion molecules on the surface of normal human monocytes. Flow-cytometric analysis showed that several subunits of the integrins, including alpha L, alpha 5, and beta 1 subunits, and intercellular adhesion molecule-1 on the monocytes were upregulated following their stimulation with 2 micrograms/ml L-MTP-PE for 24 h. Anti-alpha L antibodies blocked monocyte-mediated tumor cell killing stimulated by L-MTP-PE. We conclude that L-MTP-PE also stimulates the increase of several molecules on the monocyte cell surface. These adhesion molecules may contribute to the increased activation of monocyte-mediated tumor cell killing seen following L-MTP-PE exposure.

Identification of a combinatorial epitope expressed by the integrin alpha 4 beta 1 heterodimer involved in the regulation of cell adhesion.

The alpha 4 integrin subunit can associate with either the beta 1- or beta 7-integrin subunit to form two unique adhesion receptors alpha 4 beta 1 and alpha 4 beta 7. We developed a monoclonal antibody (mAb 19H8) that immunoprecipitated alpha 4 beta 1, induced homotypic leukocyte aggregation, and blocked the binding of cells to a synthetic peptide corresponding to the CS-1 peptide region of fibronectin. Aggregation cross-blocking analysis suggested that mAb 19H8 belonged to the group of mAbs that react with the B2 epitope of the alpha 4 subunit (alpha 4.B2 epitope); however, unlike the alpha 4.B2-specific mAb L25, mAb 19H8 did not immunoprecipitate alpha 4 beta 7. In addition, mAb 19H8 did not bind to beta 1-positive cells unless transfected with alpha 4 cDNA. These results indicated that mAb 19H8 was not specific for an individual alpha 4, beta 1, or beta 7 subunit but reacted with an epitope formed from the association of alpha 4 with beta 1. Separating the alpha 4 from the beta 1 subunit, by removing divalent cations or by treatment with high pH, disrupted mAb 19H8 binding. In contrast, the alpha 4-specific mAb L25 and the beta 1-specific mAb 18D3 could react with their respective subunits without subunit association. Therefore, mAb 19H8 defined a novel regulatory epitope expressed by the integrin alpha 4 beta 1.

Expression of a functional laminin receptor (alpha 6 beta 1, very late activation antigen-6) on human eosinophils.

The mechanisms responsible for the accumulation of eosinophils at sites of allergic and other inflammatory reactions are unknown, but recent studies have implicated both eosinophil and endothelial adhesion molecules in this process. However, less well studied have been the adhesive interactions between eosinophils and the subendothelial basement membrane and interstitial connective tissues. To test the hypothesis that eosinophils might interact with extracellular matrix proteins, we analyzed purified human eosinophils for the expression and function of various beta 1 integrins. Using indirect immunofluorescence and flow cytometry, purified eosinophils from mildly allergic donors were found to consistently express the integrin subunits beta 1 (CD29), alpha 4 (CD49d, very late activation antigen [VLA]-4 alpha), and alpha 6 (CD49f, VLA-6 alpha). No significant expression of the alpha 1, alpha 2, alpha 3, alpha 5, or beta 4 subunits was detected. Platelet contamination of the eosinophil preparations was excluded by light microscopy and by the inability to detect expression of platelet glycoproteins alpha v, CD41b, and CD42b. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of eosinophils confirmed the expression of cell-surface beta 1, alpha 4, and alpha 6. Furthermore, eosinophils purified from allergic donors were shown to adhere to plate-bound laminin, but not to type 1 or type 4 collagen. Adhesion to laminin was concentration-dependent, required divalent cations, and was completely and specifically inhibited by the anti-alpha 6 monoclonal antibody (MoAb) GoH3 and by the anti-beta 1 MoAb 33B6. Interestingly, the anti-beta 1 MoAb 18D3 (which like 33B6 blocks eosinophil binding to VCAM-1) did not inhibit eosinophil adhesion to laminin, suggesting that there are functionally distinct epitopes on the beta 1 subunit. Eosinophils purified from 4 healthy, nonallergic donors also showed alpha 6-dependent adhesion to laminin, although these cells adhered less well. These studies establish the expression of alpha 6 beta 1 on human eosinophils and document its function as a laminin receptor. Interaction of eosinophil alpha 6 beta 1 with laminin, eg, in basement membranes, may contribute to the localization of these cells at inflammatory sites in vivo.

Homotypic leukocyte aggregation triggered by a monoclonal antibody specific for a novel epitope expressed by the integrin beta 1 subunit: conversion of nonresponsive cells by transfecting human integrin alpha 4 subunit cDNA.

The monoclonal antibody 33B6 was found to be specific for the beta 1 integrin subunit. Treatment of leukocytes with this antibody induced a vigorous homotypic aggregation that had similar physiologic conditions as aggregation induced by a monoclonal antibody specific for the alpha 4 subunit. Expression of a beta 1 subunit on the cell surface was not sufficient for mAb 33B6-mediated aggregation to occur, since cells of the K562 erythroleukemia line failed to respond even though they expressed the beta 1 subunit and the 33B6 epitope. However, after transfection with cDNA encoding the alpha 4 subunit, K562 cells acquired the ability to aggregate in response to mAb 33B6 binding. By contrast, mAb 33B6 blocked cell binding to the endothelial surface protein vascular cell adhesion molecule-1 and the extracellular matrix protein fibronectin. These results suggest that the beta 1 epitope defined by mAb 33B6 may play a novel role in regulating leukocyte adhesive interactions.

Post-translational processing of the leukocyte integrin alpha 4 beta 1.

The leukocyte integrin alpha 4 beta 1 (VLA-4, CD49d/CD29) is a receptor for the extracellular matrix protein fibronectin and the endothelial adhesion protein VCAM-1. We have analyzed the biosynthesis and post-translational modifications of the two subunits of this receptor complex. The alpha 4 subunit was initially synthesized as a single-chain polypeptide that underwent the formation of complex endoglycosidase H-resistant oligosaccharide side chains and which could be proteolytically cleaved into two noncovalently associated fragments. The level and rate of alpha 4 subunit cleavage was dependent on the cell studied. The T cell tumor line HPB-ALL expressed both intact and fragmented alpha 4 on the cell surface. The interleukin-2-dependent natural killer line NK 3.3 and long term interleukin-2-dependent activated T lymphocytes cleaved the alpha 4 polypeptide earlier and more efficiently than did HPB-ALL cells and did not have detectable levels of intact alpha 4 on the cell surface. The proteolysis of alpha 4 was blocked by treating cells with either the lysosomotrophic amine NH4Cl or the carboxylic ionophore monensin. The presence of complex N-linked oligosaccharides did not seem to be necessary for alpha 4 cleavage or for binding of the alpha 4 beta 1 complex to a synthetic peptide corresponding to the binding site for this receptor on fibronectin.

Regulation of T cell proliferation by anti-CD49d and anti-CD29 monoclonal antibodies.

The beta 1 integrin VLA-4 (alpha 4 beta 1, CD49d/CD29), which is expressed on a large subpopulation of peripheral blood T lymphocytes, functions as a receptor for the endothelial adhesion protein VCAM-1 and the extracellular matrix protein fibronectin. Previous studies showed that immobilized fibronectin enhanced anti-CD3 monoclonal antibody (mAb)-induced T cell proliferation through binding to the integrins VLA-4 and VLA-5 (alpha 5 beta 1, CD49e/CD29). We studied the ability of the anti-CD49d mAb L25 to potentiate proliferation. T cell proliferation was induced by subthreshold concentrations of anti-CD3 mAb (mAb OKT3) coimmobilized with mAb L25 but not with coimmobilized anti-CD29 (beta 1) mAb. Soluble anti-CD29 mAb inhibited the proliferation induced by coimmobilized mAb OKT3 and L25 but not proliferation induced by mAb OKT3 with PMA or coimmobilized anti-CD26 mAb.

Expression and ligand-binding function of the integrin alpha 4 beta 1 (VLA-4) on neural-crest-derived tumor cell lines.

Human neural-crest-derived tumor cell lines, including three neuroblastomas, an astrocytoma, a glioblastoma, a rhabdomyosarcoma and a melanoma were screened for the expression of the integrin alpha 4 beta 1 (VLA-4). The neuroblastomas IMR-32 and SK-N-SH, the astrocytoma 131-INI, the glioblastoma Fogerty and the rhabdomyosarcoma TE-671 expressed alpha 4 beta 1 as determined by cytofluorometry and immunoprecipitation. Another neuroblastoma line, LA-N-1, did not express alpha 4 beta 1. Analysis of immunoprecipitated alpha 4 beta 1 showed that the alpha 4 subunit from the various cell types differed in relative molecular weight (M(r)). The variability in the observed M(r) could be accounted for by differences in the levels of N-linked glycosylation. The observed variability in M(r) did not appear to affect function since intact cells and solubilized alpha 4 beta 1 bound to a synthetic peptide identical in sequence to the CS-1 region of the alternatively spliced IIICS domain of fibronectin, a known alpha 4 beta 1 ligand.

Triggering of the proteinase dipeptidyl peptidase IV (CD26) amplifies human T lymphocyte proliferation.

CD26 (Ta1, dipeptidyl peptidase IV) is a Mr 105,000 protein expressed at high levels on activated T lymphocytes and is a potential marker of memory T cells. Reciprocal immunodepletion and solid phase double determinant binding studies showed that mAb AC7 and the CD26-specific mAb anti-Ta1 reacted with spatially distinct sites on the same molecule. The proteinase dipeptidyl peptidase IV (DPP IV) was immunoprecipitated with mAb AC7 and its enzymatic activity directly assayed using an enzyme overlay membrane system. High levels of DPP IV activity were detected on the T cell tumor line CCRF-HSB-2 and on PBMC stimulated by a variety of methods. By itself, soluble mAb AC7 was not mitogenic for T cells but enhanced T cell proliferation that resulted from treatment with phorbol myristic acetate (PMA) in the presence of accessory cells. T cell proliferation was also induced by co-immobilized mAb AC7 and mAb OKT3 (anti-CD3). Cultures of T cells growing in the presence of IL-2 responded with accelerated growth when exposed to a combination of immobilized mAb AC7 and soluble mAb OKT3, a result not seen with freshly isolated T cells.

Fibronectin promotes proliferation of naive and memory T cells by signaling through both the VLA-4 and VLA-5 integrin molecules.

The capacity of purified fibronectin to costimulate human T cell DNA synthesis was examined. Low concentrations of immobilized fibronectin, but not soluble fibronectin, augmented anti-CD3-induced proliferation of highly purified human T cells. In the absence of anti-CD3 stimulation, immobilized fibronectin did not induce T cell proliferation alone or in the presence of IL-2 or phorbol dibutyrate. Although fibronectin is present in high concentrations in the serum, immobilized fibronectin was able to costimulate T cell proliferation when cells were cultured in serum-containing medium. Immobilized collagen type I did not enhance anti-CD3 stimulated T cell responses, whereas gelatin (denatured collagen) and laminin were able to enhance anti-CD3 stimulated T cell responses modestly. The effects of gelatin, however, appeared to be indirect, because it could not enhance responses in medium devoid of fibronectin. Immobilized fibronectin enhanced anti-CD3 induced proliferation of both CD45RA dim and CD45RA bright subsets within both the CD4+ and CD8+ subpopulations of T cells, although cells with the CD45RA dim phenotype were costimulated by lower concentrations of immobilized fibronectin. Enhancement of anti-CD3 induced proliferation by immobilized fibronectin was completely inhibited by a mAb to CD29, the integrin beta 1-chain (4B4) and not by a variety of other mAb. In contrast to its effects on proliferation, 4B4 only partially blocked T cell binding to anti-CD3 and fibronectin-coated macrowells. These findings suggested that the interaction between fibronectin and its receptor transduced a signal to the T cell and did not merely stabilize the interaction between anti-CD3 and the CD3 complex. Further experiments confirmed this observation. Thus fibronectin could enhance anti-CD3 responses when it was immobilized to a separate surface. The augmentation of anti-CD3 stimulated proliferation induced by immobilized fibronectin was also inhibited partially by mAb to either VLA-4 or VLA-5 and completely by a combination of the two mAb. The mAb to VLA-4 not only blocked the capacity of immobilized fibronectin to enhance anti-CD3-induced T cell proliferation but also directly costimulated T cell responses. Thus, at least two fibronectin receptors are involved in fibronectin-mediated costimulation of T cell proliferation. These studies indicate that signals are transduced through the fibronectin receptors, VLA-4 and VLA-5, that augment T cell responses and therefore implicate the extracellular matrix protein fibronectin as an important influence regulating T cell responsiveness in vivo.

A monoclonal antibody to VLA-4 alpha-chain (CDw49d) induces homotypic lymphocyte aggregation.

The complex processes of cellular adhesion involve a variety of receptor to ligand interactions that are extremely important during the development of immune function. Lymphocyte activation by Ag or mitogen, CTL- and NK-mediated cytolysis, homing to lymphoid-associated tissue, and the attachment of lymphocytes to extracellular matrix proteins are all governed, at least in part, by cell surface adhesion receptors. During the analysis of mAb for the ability to block human cytotoxic T lymphocyte-mediated killing an inhibitory mAb was noted that caused rapid and vigorous aggregation among the CTL. This antibody, mAb L25, also induced aggregation among human T and B tumor cell lines. mAb L25 binds to an epitope on the alpha 4 subunit of the integrin protein VLA-4 and induced an adhesion event requiring divalent cations, energy, a fluid plasma membrane, and an intact cytoskeleton. The Ag-independent homotypic adhesion induced by mAb L25 was not inhibited by mAb to the lymphocyte function associated Ag-1 (CD11a/CD18), CD2, CD4, and CD8, or to their ligands ICAM-1, LFA-3, MHC class I, or MHC class II. We believe that these experiments suggest a role for VLA-4 in a novel system of leukocyte adhesion.

Lymphocyte surface antigen L25 is a member of the integrin receptor superfamily.

Monoclonal antibody (mAb) anti-L25 identifies an antigen on the surface of human lymphocytes. This mAb immunoprecipitated three distinct polypeptides of Mr 150,000, 85,000, and 75,000 from Nonidet P-40 lysates of surface radioiodinated lymphocytes. The three polypeptides were found under both nonreducing and reducing conditions. An additional polypeptide of Mr 130,000 was detected in mAb anti-L25 immuno-precipitates when cells were lysed with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Epitope localization experiments indicated that both the Mr 150,000 and 85,000 polypeptides contained antibody reactive sites. Peptide mapping studies demonstrated structural similarities in the Mr 150,000 and 85,000 components. The analysis of L25 subunits from lysates of antigen-stimulated T lymphocytes revealed a loss of the Mr 150,000 polypeptides in mAb anti-L25 immunoprecipitates. Solid phase double determinant binding assays demonstrated that L25 is similar and probably identical to lymphocyte surface antigen very late activation-4. This relationship placed L25 as a member of the integrin receptor superfamily.

Post-translational processing of the murine third component of complement.

The biosynthesis and secretion of the third component of complement (C3) has been studied with the macrophage cell line J774.2. C3 is initially synthesized as a single polypeptide chain precursor termed pro-C3, of relative molecular weight (Mr) 170,000 that is post-translationally modified by proteolytic cleavage into two polypeptides linked by disulphide bonds. The larger polypeptide, termed the alpha chain, has an Mr of 110,000-115,000, while the smaller beta chain has an Mr of 55,000-60,000. Pulse-chase experiments indicate that the proteolytic processing of pro-C3 occurs intracellularly, just prior to secretion. Unlike human C3, which has carbohydrate on both the alpha and beta chains, only the alpha chain of murine C3 is glycosylated. The carboxylic ionophores monensin and nigericin totally inhibit the proteolytic processing of pro-C3 at a concentration of approximately 10(-6) M. This block on proteolytic processing was shown not to be mediated by changes in intracellular pH induced by the disruption of proton gradients. Rather, data from experiments using carboxylic ionophores and other perturbants of cellular physiology indicated that the enzyme(s) responsible for the proteolytic cleavage of pro-C3 either reside in a cellular compartment with a neutral pH or are proteinases active over a relatively broad pH range.

Structural studies on the murine Ia alloantigens. VI. Evidence that both subunits of the I-A alloantigen are encoded by the I-A subregion.

The alpha and beta subunits of the murine I-A alloantigens from several H-2 haplotypes were examined by comparative tryptic peptide mapping by using double label (3H and 14C) techniques. Significant structural variation between alleles was detected in both subunits. Tryptic digests of the alpha polypeptides from s, b, and d showed only 65% co-elution with k; beta-chains from s, b, d, and r were about 50% similar to the k beta subunit. Peptide analysis of the Ak subunits from intra-H-2 recombinant strains indicated that both the alpha and beta polypeptides are encoded within the I-A subregion.