RESUMO
OBJECTIVE: CD22 is a surface molecule exclusively expressed on B cells that regulates adhesion and B cell receptor (BCR) signaling as an inhibitory coreceptor of the BCR. Central downstream signaling molecules that are activated upon BCR engagement include spleen tyrosine kinase (Syk) and, subsequently, phospholipase Cγ2 (PLCγ2), which results in calcium (Ca(2+)) mobilization. The humanized anti-CD22 monoclonal antibody epratuzumab is currently being tested in clinical trials. This study was undertaken to determine the potential mechanism by which this drug regulates B cell activation. METHODS: Purified B cells were preincubated with epratuzumab, and the colocalization of CD22 and CD79α, without BCR engagement, was assessed by confocal microscopy. The phosphorylation of Syk (Y348, Y352) and PLCγ2 (Y759) as well as the Ca(2+) flux in the cells were analyzed by flow cytometry upon stimulation of the BCR and/or Toll-like receptor 9 (TLR-9). The influence of CD22 ligation on BCR signaling was assessed by pretreating the cells with epratuzumab or F(ab')(2) fragment of epratuzumab, in comparison with control cells (medium alone or isotype-matched IgG1). RESULTS: Epratuzumab induced colocalization of CD22 and components of the BCR independent of BCR engagement, and also reduced intracellular Ca(2+) mobilization and diminished the phosphorylation of Syk and PLCγ2 after BCR stimulation in vitro. Inhibition of kinase phosphorylation was demonstrated in both CD27- and CD27+ B cells, and this appeared to be independent of Fc receptor signaling. Preactivation of the cells via the stimulation of TLR-9 did not circumvent the inhibitory effect of epratuzumab on BCR signaling. CONCLUSION: These findings are consistent with the concept of targeting CD22 to raise the threshold of BCR activation, which could offer therapeutic benefit in patients with autoimmune diseases.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Doenças Autoimunes/tratamento farmacológico , Linfócitos B/efeitos dos fármacos , Cálcio/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Adulto , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos B/citologia , Linfócitos B/metabolismo , Antígenos CD79/imunologia , Antígenos CD79/metabolismo , Relação Dose-Resposta Imunológica , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Imunoglobulina G/farmacologia , Masculino , Pessoa de Meia-Idade , Fosfolipase C gama/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais/imunologia , Receptor Toll-Like 9/metabolismo , Adulto JovemRESUMO
Although the in vivo interaction between polymorphonuclear neutrophils (PMN) and fibroblasts may be important, these pathways have not been well studied. We have investigated the adherence of PMN to monolayers of human fetal lung fibroblasts, using a microtiter plate assay based upon the uptake by cells of the vital stain Rose Bengal. Stimulation with phorbol myristate acetate (PMA) caused a significant increase of adherence over basal levels which was rapid in onset and plateaued at 5 min. Adhesion was dependent on the leucocyte integrin family of glycoproteins, notably on Mac-1, since monoclonal antibodies toward the beta chain (CD18) and alpha chain (CD11b) of Mac-1 almost completely suppressed PMA-induced PMN adhesion (88% and 77% inhibition, respectively). Adhesion was also inhibited by the peptides RGDS and GRGDS (24.2% and 26.6%, respectively using 1 mM peptide). Prestimulation of fibroblasts for longer time periods (5 and 24 h) with interleukin 1 alpha and tumor necrosis factor alpha, but not transforming growth factor beta, also resulted in a significant increase in adhesion of unstimulated PMN (after 24 h preincubation, 10 U/ml IL1 alpha stimulated adhesion by 179% of control, 500 U/ml TNF alpha by 157%). This indicated that there are both PMN- and fibroblast-dependent pathways for PMN adhesion. Components of the extracellular matrix of fibroblasts do not appear to play important roles in the adhesion process since addition of fibronectin and type IV collagen, or of purified antibodies to fibronectin and types I and IV collagen, did not affect PMA-induced PMN adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Adesão Celular/fisiologia , Fibroblastos/fisiologia , Neutrófilos/fisiologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/fisiologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Integrinas/fisiologia , Interleucina-1/farmacologia , Cinética , Pulmão/citologia , Dados de Sequência Molecular , Neuraminidase/metabolismo , Neutrófilos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
1. Although anti-alpha(4) integrin mAbs reduce eosinophil accumulation in several models of allergic inflammation, it is not clear whether this occurs via a direct action to block eosinophil alpha(4) integrins or indirectly on another cell type. The role of alpha(4) integrins on the accumulation of (111)In-labelled eosinophils in allergic and non-allergic inflammation in guinea-pig skin was therefore investigated. 2. Intradermal injection of antigen in sensitized skin sites induced accumulation of (111)In-eosinophils that was reduced up to 70% by two anti-alpha(4) integrin mAbs. In contrast, accumulation of (111)In-eosinophils to intradermal chemoattractants was unaffected by the same mAbs. 3. Accumulation of (111)In-eosinophils in allergic and non-allergic conditions was partly inhibited by a low dose of an anti-beta(2) integrin mAb. In combination with anti-alpha(4) integrin mAb, responses were not further reduced suggesting that these adhesion pathways are not additive or synergic. 4. Pretreating skin sites with antiserum or contaminating LPS did not reveal an alpha(4) integrin dependent pathway for chemoattractant-induced (111)In-eosinophil accumulation. These data suggest that alpha(4) integrins are involved in the response to antigen in sensitized skin sites. 5. Pretreating (111)In-eosinophil with alpha(4) integrin mAb blocked their adhesion to fibronectin in vitro but did not inhibit their accumulation in allergic inflammation suggesting that the blocking effect in vivo was eosinophil independent. 6. These data support the concept that targeting alpha(4) integrins on cells other than eosinophils could control eosinophil accumulation and have therapeutic potential in allergic diseases such as asthma and atopic dermatitis.
Assuntos
Antígenos CD/metabolismo , Eosinófilos/fisiologia , Hipersensibilidade/metabolismo , Inflamação/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Citometria de Fluxo , Cobaias , Humanos , Indicadores e Reagentes , Radioisótopos de Índio , Integrina alfa4 , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Pele/citologia , Zimosan/farmacologiaRESUMO
1. Intradermal injection of the complement fragment C5a des Arg induces local oedema formation that, in rabbits and man, is dependent on circulating neutrophils. Monoclonal antibodies to the leukocyte adhesion molecule CD11/CD18 block neutrophil accumulation and prevent neutrophil-dependent oedema formation. The role of CD11/CD18 in mediating eosinophil accumulation in vivo is less established. In this study we have used an anti-human CD18 monoclonal antibody, 6.5E, to investigate the neutrophil-dependency of oedema formation induced by C5a des Arg in guinea-pig skin. We also studied the role of CD18 in mediating eosinophil accumulation in the same model. 2. Stimulated adhesion of 111In-labelled guinea-pig neutrophils and eosinophils to serum-coated plastic was inhibited in a dose-dependent manner by 6.5E suggesting that the monoclonal antibody recognizes and blocks the guinea-pig CD18 adhesion molecule. 3. The accumulation of 111In-labelled neutrophils induced by zymosan-activated plasma (ZAP, as a source of C5a des Arg) in skin sites was reduced by up to 89% in animals treated intravenously with F(ab')2 fragments of 6.5E. ZAP-induced accumulation of 111In-labelled eosinophils was also greatly reduced (by up to 78%) by treatment with 6.5E. 4. Despite the inhibition of ZAP-induced neutrophil accumulation by 6.5E, local oedema formation in the same skin sites was unaffected, except at the top dose of ZAP, by treatment with the anti-CD18 monoclonal antibody, suggesting that the oedema response was largely neutrophil-independent. Indeed, ZAP-induced oedema formation was reduced by up to 81% by the H1 receptor antagonist, mepyramine. 5. Accumulation of 111 In-labelled eosinophils in a passive cutaneous anaphylactic (PCA) reaction was also blocked by treatment with 6.5E, while oedema formation in the same skin sites was unaffected.Intradermal injection of cationic protein-containing extracts of Schistosoma mansoni larvae also induced the accumulation of 111 In-labelled neutrophils and eosinophils which was abrogated by intravenous 6.5E.In contrast, extract-induced local oedema formation was similar in control and 6.5E-treated guinea-pigs.6. In summary, the local accumulation of radiolabelled neutrophils at sites of inflammation in guinea pigskin was dependent on the adhesion molecule CD18 while, in contrast, there was no evidence for neutrophil-dependent oedema formation in this species. Accumulation of radiolabelled eosinophils was also dependent on CD18.
Assuntos
Antígenos CD/fisiologia , Dermatite/patologia , Edema/patologia , Eosinófilos/fisiologia , Neutrófilos/fisiologia , Animais , Anticorpos Monoclonais , Antígenos de Bactérias/imunologia , Antígenos CD/imunologia , Antígenos CD18 , Adesão Celular/efeitos dos fármacos , Complemento C5a des-Arginina , Eosinófilos/citologia , Feminino , Cobaias , Humanos , Fragmentos Fab das Imunoglobulinas , Radioisótopos de Índio , Teste de Inibição de Aderência Leucocítica , Masculino , Camundongos , Mycobacterium bovis/imunologia , Neutrófilos/citologia , Anafilaxia Cutânea Passiva/fisiologia , Coelhos , Schistosoma mansoni , Zimosan/farmacologiaRESUMO
Triggered polymorphonuclear leucocytes (PMNL) can decrease the elastase inhibitory capacity of serum by inactivating the main inhibitor of elastase alpha-1-proteinase inhibitor (alpha-1-PI). Maximal inactivation occurs with stimuli that release myeloperoxidase from PMNL along with hydrogen peroxide. Specific protection of alpha-1-PI function is obtained with antioxidants that interfere with this system. PMNL that are activated with phorbol myristate acetate release hydrogen peroxide but not myeloperoxidase, and only inactivate alpha-1-PI in the presence of exogenously-added PMNL-derived supernatants which contain this enzyme. Cell-free inactivation requires both active enzyme and hydrogen peroxide, and is greatest at pH 6.2, the pH optimum for myeloperoxidase-catalysed inactivation of alpha-1-PI. This data supports the notion that leucocyte myeloperoxidase may act to suppress the antiprotease screen afforded by alpha-1-PI by generating hypochlorous acid in the presence of chloride and respiratory burst-derived hydrogen peroxide, and in the microenvironment of lowered pH associated with degranulation. Pulmonary emphysema seems to be associated with an imbalance between elastase and its inhibitors at the lung surface. PMNL are likely to play an important role in the pathogenesis of emphysema since they contain both elastase, which can solubilize connective tissue elastin, and the constituents of an oxidative system which can inactivate the most important antielastase, alpha-1-PI.
Assuntos
Proteínas Sanguíneas , Neutrófilos/metabolismo , Peroxidase/metabolismo , Antioxidantes/farmacologia , Sistema Livre de Células , Citocalasina B/farmacologia , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/enzimologia , Consumo de Oxigênio/efeitos dos fármacos , Frações Subcelulares/enzimologia , Acetato de Tetradecanoilforbol/farmacologia , alfa 1-AntitripsinaRESUMO
Unactivated peripheral blood leukocytes show little tendency to bind to other cells or matrix components, whilst, in the presence of inflammatory mediators, adhesive interactions can rapidly increase. The Leu-CAM (beta 2 integrin) family of adhesion molecules have been shown to mediate a variety of these induced adhesion events. Here we describe a monoclonal antibody against CD18, KIM185, which stimulates JY cell homotypic aggregation by a CD11 a pathway as well as inducing the adherence of neutrophils to protein-coated plastic by a CD11b-dependent mechanism. The antibody recognizes an epitope distinct from the previously described KIM127 antibody and evidence is presented that the binding of KIM185 can cause a change in the conformation of the CD18 molecule.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/química , Adesão Celular , Antígeno-1 Associado à Função Linfocitária/fisiologia , Antígeno de Macrófago 1/fisiologia , Antígenos CD/imunologia , Antígenos CD/fisiologia , Antígenos CD11 , Antígenos CD18 , Linhagem Celular , Complemento C3b/metabolismo , Epitopos , Humanos , Conformação ProteicaRESUMO
Elastin in vivo is likely to be complexed with amphipathic ligands such as lipids. The susceptibility of stable [3H] elastin-fatty acid complexes to the action of porcine pancreatic elastase (PPE) and to human neutrophil lysates over time was assessed. Elastolysis by PPE of substrates prepared with oleic or linoleic acids was initially higher (for up to 2 hours) than that of uncomplexed elastin. Stearic acid and elaidic acid (the trans isomer of oleic acid) did not enhance the elastolytic rate above control. The stimulatory effect of oleic and linoleic acids appeared to derive from increased adsorption of PPE onto elastin; the loss of stimulatory activity over time occurred in parallel with a progressive decrease in adsorption. All fatty acids tested inhibited elastolysis by neutrophil lysates, the effect being particularly marked with oleic and elaidic acids. These results indicate that 1). Complexed fatty acids can modulate the rate of elastin breakdown by elastases; 2). The effects observed with PPE are due to differences in adsorption of enzyme onto substrate, possibly as a result of steric considerations; 3). Since elastolysis by neutrophil lysates is inhibited by all fatty acids, the properties of different elastolytic enzymes should be considered in in vitro model systems of connective tissue breakdown.
Assuntos
Elastina/metabolismo , Ácidos Graxos/metabolismo , Elastase Pancreática/metabolismo , Adsorção , Animais , Bovinos , Humanos , Técnicas In Vitro , Neutrófilos/metabolismo , Especificidade por SubstratoRESUMO
Connective tissue formation at sites of tissue repair is regulated by matrix protein synthesis and degradation, which in turn is controlled by the balance between proteases and antiproteases. Recent evidence has suggested that antiproteases may also exert direct effects on cell function, including influencing cell migration and proliferation. The antiprotease, alpha1-antitrypsin, is the major circulating serine protease inhibitor which protects tissues from neutrophil elastase attack. Its deficiency is associated with the destruction of connective tissue in the lung and the development of emphysema, whereas accumulation of mutant alpha1-antitrypsin within hepatocytes often leads to liver fibrosis. In this study, we report that alpha1antitrypsin, at physiologically relevant concentrations, promotes fibroblast proliferation, with maximal stimulatory effects of 118 +/- 2% (n=6, P < 0.02) above media controls for cells exposed to 60 microM. We further show that alpha1antitrypsin also stimulates fibroblast procollagen production, independently of its effects on cell proliferation, with values maximally increased by 34 +/- 3% (n = 6, P < 0.01) above media controls at 30 microM. Finally, mechanistic studies to examine the mechanism by which alpha1-antitrypsin acts, showed that alpha1-antitrypsin induced the rapid activation of p42MAPK and p44MAPK (also known as ERK1/2) and that the specific MEK1 inhibitor PD98059 totally blocked alpha1-antitrypsin's mitogenic effects. These results support the hypothesis that alpha1-antitrypsin may play a role in influencing tissue repair in vivo by directly stimulating fibroblast proliferation and extracellular matrix production via classical mitogen-activated signalling pathways.
Assuntos
Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Plantas/farmacologia , Pró-Colágeno/biossíntese , Inibidores de Serina Proteinase/farmacologia , Transdução de Sinais/efeitos dos fármacos , alfa 1-Antitripsina/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Proteínas Tirosina Quinases/fisiologia , Inibidores da Tripsina , alfa-Amilases/antagonistas & inibidoresRESUMO
Chronic inhalation of cadmium fumes has been associated with the development of emphysema, a disease characterized by extensive disruption of lung connective tissue. Cadmium is also an important contaminant of tobacco and may play a role in cigarette smoking-related emphysema. In this paper we investigated the effect of nontoxic doses of cadmium chloride (CdCl2) on fibroblast procollagen production and proliferation, key features of connective tissue repair following injury. CdCl2 inhibited fibroblast procollagen production in a dose-dependent manner in two different cell lines. For fetal rat fibroblasts, maximal effects were observed at 10 microM CdCl2, with values reduced by 82 +/- 6% (mean +/- SE, n = 6, P < 0.01) relative to control cells. In contrast, noncollagen protein synthesis by these cells was enhanced in the presence of CdCl2. In human fetal lung fibroblasts (HFL1), maximal inhibition of procollagen production (83 +/- 2%, P < 0.01) was observed at 40 microM CdCl2, whereas noncollagen protein synthesis was unaffected. In both cell lines the inhibition of procollagen production was shown to be due to decreased procollagen synthesis and an increase in the proportion of newly synthesized procollagen degraded. Cadmium also affected fibroblast proliferation in response to 2% serum, with values for fetal rat cells depressed by 17 +/- 4, 72 +/- 2, and 86 +/- 4% (all P < 0.01) compared with controls at 1, 5, and 10 microM CdCl2, respectively. These data show that cadmium selectively inhibits fibroblast procollagen production and also attenuates their mitogenic response to serum.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cádmio/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pró-Colágeno/antagonistas & inibidores , Animais , Cloreto de Cádmio , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cloretos/farmacologia , Compostos Ferrosos/farmacologia , Feto/citologia , Feto/metabolismo , Fibroblastos/citologia , Humanos , Cinética , Pulmão/citologia , Pulmão/embriologia , Pró-Colágeno/biossíntese , Biossíntese de Proteínas , RatosRESUMO
The integrin adhesion molecules are involved in the recruitment and activation of inflammatory cells at sites of inflammation in a variety of diseases. In the present study, we have investigated the effects of blocking monoclonal antibodies (mAbs) directed against CD49d (alpha(4) integrin), CD18 (beta(2) integrin) and the alpha sub-units of beta(2) integrin CD11a (LFA-1 integrin) and CD11b (Mac-1 integrin), on antigen (Ag)-induced acute bronchoconstriction and cellular recruitment in allergic rabbits in vivo. Inhaled Ag (Alternaria tenuis) challenge of neonatally sensitised rabbits caused an acute bronchoconstriction demonstrated by an increase in lung resistance (R(L)) and decrease in dynamic compliance (C(dyn)) and pulmonary inflammation characterised by an increase in bronchoalveolar lavage (BAL) inflammatory cells, particularly eosinophils, 24 h after challenge. Pre-treatment with the anti-CD49d mAb (Max-68P), significantly inhibited the Ag-induced acute bronchoconstriction in terms of R(L) and (C(dyn)). Treatment with the other anti-integrin mAbs had no effect on the acute bronchoconstriction after inhaled Ag challenge.Pre-treatment with the anti-integrin mAbs had differential effects in blocking the recruitment of inflammatory cells 24 h after inhaled Ag in the allergic rabbits. The data show that in the allergic rabbit model of asthma, VLA-4 (CD49d/CD29) only, is involved in the acute bronchoconstriction, suggesting an involvement of mast cell degranulation. Furthermore, eosinophil recruitment and activation appears to be mediated by a combination of VLA-4 (CD49d/CD29) and LFA-1 (CD18/CD11a). However in contrast, lymphocyte recruitment appears to be mediated by a combination of LFA-1 (CD18/CD11a) and Mac-1 (CD18/CD11b).
Assuntos
Obstrução das Vias Respiratórias/imunologia , Anticorpos Monoclonais/fisiologia , Moléculas de Adesão Celular/efeitos adversos , Moléculas de Adesão Celular/imunologia , Integrinas/antagonistas & inibidores , Integrinas/imunologia , Mastócitos/metabolismo , Pneumonia/etiologia , Pneumonia/imunologia , Obstrução das Vias Respiratórias/etiologia , Alternaria/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Fungos/efeitos adversos , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Broncoconstrição/imunologia , Antígeno CD11a/efeitos dos fármacos , Antígeno CD11a/imunologia , Antígeno CD11b/efeitos dos fármacos , Antígeno CD11b/imunologia , Antígenos CD18/efeitos dos fármacos , Antígenos CD18/imunologia , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Peroxidase de Eosinófilo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Integrina alfa4/efeitos dos fármacos , Integrina alfa4/imunologia , Integrina alfa4beta1/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Mastócitos/imunologia , Peroxidases/química , Coelhos , Transducina/antagonistas & inibidoresRESUMO
The remodeling of pulmonary vessels that occurs in association with pulmonary hypertension involves, in part, thickening of the adventitia. The stimulus for this process is not understood. One explanation is that endothelial cells secrete a growth factor that expands the local population of fibroblasts by acting as a chemoattractant and mitogen. Endothelins are a family of potent newly discovered vasoactive peptides. One of these compounds, endothelin-1 (ET-1), is secreted by endothelial cells and is known to constrict pulmonary vessels. Another, endothelin-3 (ET-3), is not secreted by endothelial cells and is less potent as a pulmonary vasoconstrictor. We hypothesized that the endothelins may have the capacity both to constrict these vessels and to initiate fibroblast chemotaxis and replication. Here we investigated the effects of both ET-1 and ET-3 on the chemotaxis and replication of fibroblasts derived from pulmonary vessels. Cells were isolated from rat pulmonary arteries, cultured in medium and 10% newborn calf serum, and used between passages 2 and 5. Chemotaxis was assessed using a modified Boyden chamber with a polycarbonate filter (pore size, 8 microns) separating cells in the upper chambers from endothelin in the lower chambers. Replication was assessed both by direct cell counts and by a colorimetric assay based on uptake and subsequent release of methylene blue. Both ET-1 and ET-3 induced chemotaxis of pulmonary artery fibroblasts and did so in a dose-dependent fashion. The maxima for both peptides occurred at a concentration of about 10(-7) M, when chemotaxis was greatest for ET-1 (22 +/- 1.4 versus 14 +/- 1.8 cells/grid [mean +/- SEM], (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Divisão Celular/efeitos dos fármacos , Quimiotaxia , Endotelinas/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Masculino , Artéria Pulmonar/citologia , Ratos , Vasoconstrição/efeitos dos fármacosRESUMO
A series of fusion proteins have been generated between human and mouse CD18. These proteins have been used to carry out preliminary mapping studies on a number of anti-CD18 antibodies including KIM127 an antibody that promotes CD18-dependent adhesion. This antibody maps to a region of the CD18 molecule between amino acids 406 and 570 in a region containing cysteine-rich repeats.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD18/imunologia , Adesão Celular/imunologia , Cisteína/genética , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos , Western Blotting , Antígenos CD18/química , Antígenos CD18/genética , Linhagem Celular/fisiologia , Cisteína/análise , Mapeamento de Epitopos , Expressão Gênica/genética , Expressão Gênica/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Fatores de TempoRESUMO
Beta 2-integrins play a crucial role in the development of an inflammatory response. In ours study, Abs have been used to investigate the role of individual members of this family of adhesion molecules in both in vivo and in vitro assays. An Ab against rabbit LFA-1 effectively inhibited the adhesion of rabbit polymorphonuclear leukocytes to rabbit endothelial cells in culture and was also effective in blocking cell recruitment to the peritoneum and vascular leakage at dermal sites of inflammation. An Ab that inhibited rabbit complement receptor type 3 function in vitro failed to inhibit cell recruitment to the peritoneum or vascular leakage in response to intradermal FMLP. Histologic studies suggested that the anti-complement receptor type 3 Ab may have modified the cell migration process.
Assuntos
Antígenos CD18/fisiologia , Adesão Celular , Antígeno-1 Associado à Função Linfocitária/fisiologia , Antígeno de Macrófago 1/fisiologia , Neutrófilos/fisiologia , Animais , Anticorpos Monoclonais , Bradicinina/farmacologia , Permeabilidade Capilar , Células Cultivadas , Dermatite/fisiopatologia , Técnicas In Vitro , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , CoelhosRESUMO
Eosinophils have been implicated in several disorders associated with the development of fibrosis. This led us to investigate the interactions between eosinophils and fibroblasts in vitro. Adhesion between purified guinea pig peritoneal eosinophils and monolayers of human fetal lung fibroblasts was assessed using the rose bengal dye staining assay. Fibroblast replication was assessed using a colorimetric assay based upon the uptake and subsequent release of methylene blue. Addition of phorbol myristate acetate induced a rapid, time-dependent increase in eosinophil adhesion (127% and 328% over basal adhesion after 10 and 30 min, respectively). Phorbol myristate acetate-induced adhesion was inhibited by the peptides RGDS and GRGDS (48% and 42%, respectively using 1 mM peptide) and by nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism (46% inhibition at 15 microM). In addition, 24 h culture of fibroblast monolayers with interleukin 1 alpha (IL-1 alpha) or tumour necrosis factor alpha (TNF alpha) resulted in enhanced adhesion (10 U/ml IL-1 alpha stimulated adhesion by 55% of control, 500 U/ml TNF alpha by 75% of control). Conditioned media from cultured eosinophils stimulated fibroblast replication in a time-dependent fashion with maximal stimulation at 3 h. In contrast, media from guinea pig peritoneal macrophages in culture did not show such an effect. This study indicates that eosinophils are capable of both adhering to and releasing mitogens for fibroblasts in vitro. These observations suggest that eosinophils have the capacity to play a role in the development of fibrosis in disorders where they have been shown to be present.
Assuntos
Eosinófilos/fisiologia , Fibroblastos/citologia , Pulmão/citologia , Sequência de Aminoácidos , Animais , Adesão Celular , Divisão Celular , Células Cultivadas , Eosinófilos/citologia , Cobaias , Humanos , Técnicas In Vitro , Interleucina-1/farmacologia , Masculino , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Peptídeos/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Acyclic, disulphide derivatives of cysteine have been identified as moderately potent antagonists of alpha4beta1-mediated leukocyte cell adhesion to VCAM. This communication describes how they were discovered from a simple L-cystine derivative and using the structure-activity data of C*DThioPC* related cyclic peptides.
Assuntos
Cisteína/química , Integrinas/antagonistas & inibidores , Receptores de Retorno de Linfócitos/antagonistas & inibidores , Cisteína/farmacologia , Integrina alfa4beta1 , Interleucina-8/farmacologia , Ligação Proteica , Relação Estrutura-Atividade , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Using disulphide cysteine-based inhibitors as lead structures, this communication describes our strategy for identifying more stable, potent antagonists of the alpha4beta1 integrin. These studies ultimately discovered potent, low molecular weight inhibitors based on D-thioproline-L-tyrosine.