RESUMO
Latent membrane protein 1 (LMP1) plays a critical role in B cell transformation by Epstein-Barr virus (EBV) and appears to mimic a constitutively active CD40 receptor. Intracellular tumor necrosis factor (TNF) receptor-associated factor (TRAF) adapter proteins, shown to contribute to signaling by both CD40 and LMP1, were recruited by both molecules to lipid-enriched membrane rafts. However, we found that TRAFs 2 and 3 were subsequently degraded after CD40- but not LMP1-induced signaling. This degradation was proteasome-dependent and required direct TRAF binding by CD40. Using a model system designed to directly compare the signaling potency of the cytoplasmic domains of LMP1 and CD40 in B lymphocytes, we found that LMP1 more potently activates c-Jun kinase and nuclear factor kappaB and induces higher levels of several B cell effector functions than does CD40. This suggests that LMP1 utilizes a modified CD40 signaling pathway. Failure to regulate TRAFs may contribute to the enhanced capacity of LMP1 to activate B cells as well as promote B cell transformation.
Assuntos
Linfócitos B/imunologia , Antígenos CD40/fisiologia , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Proteínas da Matriz Viral/metabolismo , Animais , Linfócitos B/virologia , Antígenos CD40/genética , Linhagem Celular , Membrana Celular/imunologia , Herpesvirus Humano 4/fisiologia , Humanos , Ativação Linfocitária , Camundongos , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF , Fator 3 Associado a Receptor de TNF , TransfecçãoRESUMO
T cell dependent B lymphocyte activation requires interactions between numerous receptor-ligand pairs on the two cell types. Recently, advances have been made both in understanding how these various signals regulate B cell effector functions and in identifying many new receptor-ligand pairs that contribute to the regulation of B cell function by T lymphocytes.
Assuntos
Linfócitos B/imunologia , Comunicação Celular/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Animais , Linfócitos B/metabolismo , Adesão Celular/imunologia , Cooperação Linfocítica , Linfócitos T/metabolismoRESUMO
We have recently found (O. W. Rokhlin et al., Cancer Res., 57: 1758-1768, 1997) that, although Fas ligation induced apoptosis in two of six human prostatic carcinoma cell lines investigated, the apoptotic machinery involved in Fas-mediated killing is already in place in Fas-resistant cell lines. Here, we investigated Fas- and tumor necrosis factor-alpha (TNF-alpha)-mediated apoptosis in cell hybrids between resistant (DU145 and JCA1) and sensitive (ALVA31 and PC3) cell lines. All three types of hybrid cells investigated, F1(DU145 x PC3), F1(JCA1 x PC3), and F1(JCA1 x ALVA31), were found to be resistant to Fas- and TNF-alpha-mediated apoptosis at the same level as the corresponding parental resistant cell lines. These results indicate that resistance to Fas- and TNF-alpha-mediated apoptosis dominates over sensitivity in cell hybrids and suggest that resistance may be regulated by an apoptosis suppressor factor or factors acting in resistant but not in sensitive cells.
Assuntos
Apoptose , Carcinoma/patologia , Neoplasias da Próstata/patologia , Fator de Necrose Tumoral alfa/fisiologia , Receptor fas/fisiologia , Divisão Celular , Fragmentação do DNA , Genes Dominantes , Humanos , Células Híbridas , Imunofenotipagem , MasculinoRESUMO
Of six prostatic carcinoma cell lines examined (ALVA31, DU145, JCA1, LNCaP, ND1, and PC3) by flow cytometric analysis, all were found to be positive for Fas antigen. Furthermore, of the prostate tissue specimens studied (six cases), all revealed Fas expression in benign and malignant epithelial cells. The agonistic anti-Fas monoclonal antibody (IPO-4) induced apoptosis in only two of six cell lines investigated, PC3 and ALVA31. PCR analysis indicated that all cell lines expressed normal transmembrane and death domains of Fas antigen. Using Western blot analysis, we found abundant expression of p53 in the cytoplasm of two Fas-resistant cell lines, DU145 and ND1, and did not find p53 in two Fas-sensitive cell lines, PC3 and ALVA31. Western blot and PCR analysis did not show consistent differences between cell lines examined in the expression of Bcl-2, Bcl-X(L), Bcl-X(S), and Bak. In contrast, Bax protein was not detected in two Fas-resistant cell lines, DU145 and ND1. We also showed that three Fas-resistant cell lines, DU145, ND1, and JCA1, expressed CD40, whereas the two Fas-sensitive cell lines, PC3 and ALVA31, were CD40 negative. Fas-sensitive cell lines were transfected with the cDNA encoding CD40, and the CD40-positive transfectant became more resistant to growth inhibition mediated by treatment with TNF-alpha and anti-Fas monoclonal antibody. Treatment with cycloheximide converted the phenotype of resistant cell lines from Fas resistant to Fas sensitive. Moreover, anti-Fas treatment of both resistant and sensitive cell lines induced rapid tyrosine phosphorylation or dephosphorylation of multiple proteins. These results suggest that the apoptotic machinery involved in DNA fragmentation is already in place in Fas-resistant cell lines, and thus, Fas-mediated apoptosis could be a target for therapeutic intervention.
Assuntos
Apoptose , Neoplasias da Próstata/patologia , Receptor fas/fisiologia , Reações Antígeno-Anticorpo , Antígenos CD40/genética , Antígenos CD40/metabolismo , Divisão Celular , Cicloeximida/farmacologia , Fragmentação do DNA , Citometria de Fluxo , Humanos , Masculino , Fosfotirosina/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
We recently reported that the mannan component of Trichophyton rubrum cell wall (TRM) has an inhibitory influence on cell-mediated immune function in vitro. We now describe experiments designed to identify the target cell for this effect of TRM. T. rubrum mannan labeled with fluorescein (FITC-TRM) was incubated with peripheral blood mononuclear leukocytes, monocytes, or lymphocytes. Binding and uptake of the FITC-TRM were monitored by fluorescence microscopy and flow cytometry. Approximately 10% of mononuclear leukocytes were stained with this reagent and the fluorescent cells appeared to be monocytes by morphology. Virtually all purified monocytes and no purified lymphocytes stained with FITC-TRM. Flow cytometry to analyze FITC-TRM monocyte-specific binding of FITC-TRM involved the use of a phycoerythrin-labeled anti-CD14 antibody to identify monocytes. The only cells stained with FITC-TRM were those stained with the monocyte-specific antibody. The ability of monocytes to endocytose mannan was assessed by fluorescence microscopy. Cells were exposed to FITC-TRM and washed, and the staining pattern recorded periodically over a 48-h incubation period. After 15 min, staining was homogeneous and involved the entire cell surface; by 30 min, "patching" was observed; by 90 min, bright granules had formed along the cell border and a large number of small granules were present in the cytoplasm; by 8-12 h, the fluorescent granules were enlarged in size and reduced in number; by 24-36 h, the intensity of cytoplasmic fluorescence began to diminish; and, after 48 h, all fluorescent staining had disappeared. An additional feature of staining during the 8-12-h period was the appearance of a large round bright spot in the nuclear region of each cell, which may represent nucleolar staining. A role for "mannan receptors" is suggested by observations that FITC-TRM binding was prevented by unlabeled TRM or pretreatment of the monocytes with trypsin. Our finding that monocytes selectively and specifically bind TRM appears to identify the monocyte rather than the lymphocyte as the target cell for the inhibitory effect of mannan on cell-mediated immune function.
Assuntos
Mananas/metabolismo , Monócitos/metabolismo , Trichophyton/metabolismo , Adulto , Células Cultivadas , Endocitose , Citometria de Fluxo , Fluoresceína , Fluoresceínas , Humanos , Pessoa de Meia-Idade , Receptores de Superfície Celular/metabolismo , Trichophyton/imunologiaRESUMO
CD40 is a member of the growing tumor necrosis factor receptor (TNF-R) family of molecules, and has been shown to play important roles in T cell-mediated B lymphocyte activation. Ligation of B cell CD40 by CD154 expressed on activated T cells stimulates B cell proliferation, differentiation, isotype switching, upregulation of surface molecules contributing to antigen presentation, development of the germinal center, and the humoral memory response. Several distinct structural motifs in the CD40 cytoplasmic domain regulate various CD40 signaling pathways, which involve both the TNF-R associated factors (TRAFs) and additional signaling proteins, and lead to activation of kinases and transcription factors. CD40-mediated B cell activation is mimicked by several biological response modifiers, as well as by a viral oncoprotein encoded by the Epstein-Barr virus (EBV).
Assuntos
Linfócitos B/imunologia , Antígenos CD40/metabolismo , Mimetismo Molecular , Sequência de Aminoácidos , Transformação Celular Neoplásica , Ativação Linfocitária , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de SinaisRESUMO
CD40, a member of the growing tumor necrosis factor receptor (TNF-R) family of molecules, functions as a transmembrane signal receptor in both hematopoietic and non-hematopoietic cell types, although its physiological roles are less well understood in the latter. Much has been learned over the past decade about the role of CD40 signaling in various cellular functions. In addition, some of the molecular events which occur subsequent to CD40 engagement have been characterized, although much remains to be understood. This review will summarize the known important biological roles of CD40, and discuss what is currently known about how CD40 signals.
Assuntos
Antígenos CD40/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Membrana Celular/imunologia , Ativação Enzimática , Humanos , Fosfotransferases/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
In B lymphocytes, CD40 signals contribute to the activation of Ab secretion, isotype switching, T cell costimulation, and immunological memory. TRAF proteins appear to be important components of the CD40 signal transduction complex, but their roles in the activation of B cell effector functions are poorly understood. We examined the contributions of TNF receptor-associated factor 2 (TRAF2) and TRAF3 to CD40-activated differentiation in mouse B cells transfected with inducible TRAF and dominant-negative TRAF cDNAs. We find that binding of TRAF2 and TRAF3 to CD40 is not required for the induction of Ab secretion, but that both TRAF molecules can regulate the activation process. We demonstrate a negative regulatory role for TRAF3 and that this activity is dependent on the availability of an intact TRAF3-binding site in the cytoplasmic domain of CD40. In contrast, TRAF2 appears to play a positive role in B cell differentiation, and this activity is apparent even when its binding site on CD40 is disrupted.
Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Antígenos CD40/fisiologia , Proteínas/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Linfócitos B/metabolismo , Antígenos CD40/genética , Antígenos CD40/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Humanos , Ativação Linfocitária , Camundongos , Dados de Sequência Molecular , Ligação Proteica/imunologia , Biossíntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Fator 2 Associado a Receptor de TNF , Fator 3 Associado a Receptor de TNF , Transfecção/imunologia , Células Tumorais CultivadasRESUMO
Following infection of adult mice with lymphocytic choriomeningitis virus (LCMV) there is a well documented suppression of T-cell and B-cell functions concurrent with the strong anti-LCMV immune response. Macrophages have been shown to be infected and activated during acute LCMV infection and there is some evidence to indicate that there is altered antigen presentation in acutely infected mice. We have examined nitric oxide (NO) production by splenic macrophages during acute infection of adult mice. Our results show that these macrophages are primed for production of NO, that the inducible production of NO parallels the immune suppression, and that NO production is dependent on the presence of IFN gamma. However, neither in vivo nor in vitro treatment with N-monomethyl-L-arginine (NMA), a specific inhibitor of nitric oxide synthase, altered the induction or maintenance of virus-induced immune suppression in mice acutely infected with LCMV.
Assuntos
Tolerância Imunológica/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Divisão Celular , Células Cultivadas , Concanavalina A/farmacologia , Feminino , Interferon gama/metabolismo , Interferon gama/fisiologia , Interleucina-2/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/antagonistas & inibidores , Baço/citologia , Linfócitos T/citologia , Linfócitos T/metabolismo , ômega-N-MetilargininaRESUMO
CD40 signaling to B cells is important for generating an effective humoral immune response. CD40 ligation leads to B cell activation events such as proliferation, Ig secretion, isotype switching, and up-regulation of cell surface molecules, as well as the generation of memory B cells. Many of these events are dependent upon the ability of CD40 to activate the transcription factor NF-kappa B (NF-kappa B). To define the CD40 signaling components upstream of NF-kappa B activation and the functional consequences downstream of NF-kappa B activation, we examined mouse B cell transfectants expressing wild-type or mutant human CD40. Analysis of CD40 cytoplasmic domain truncation and point mutants defined a 10-amino acid CD40 cytoplasmic signaling determinant required for NF-kappa B activation. A threonine residue at position 234, previously shown to be important for CD40 association with TNF receptor-associated factor 2 (TRAF2), TRAF3, and TRAF5, was not required for NF-kappa B activation. This suggests that in B cells, CD40-induced NF-kappa B activation can occur independently of TRAF2 and TRAF5 association. NF-kappa B activation was independent of the transmembrane domain of CD40, suggesting that it is independent of p23, a molecule that associates with CD40 in a region other than the cytoplasmic domain. Proteasome-dependent inhibitory kappa B alpha (I kappa B alpha) and I kappa B beta degradation occurred downstream of CD40 ligation and preceded CD40-mediated NF-kappa B nuclear translocation. CD40- or pervanadate-mediated I kappa B tyrosine phosphorylation was not detected. NF-kappa B activation correlated with the ability of CD40 to induce Ab secretion and the up-regulation of ICAM-1 and LFA-1. However, NF-kappa B activation was insufficient for CD40-mediated up-regulation of B7-1, Fas, and CD23.
Assuntos
Linfócitos B/imunologia , Antígenos CD40/química , Antígenos CD40/fisiologia , Epitopos de Linfócito B/química , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Transporte Biológico/efeitos dos fármacos , Antígenos CD40/genética , Citoplasma/química , Citoplasma/imunologia , Citoplasma/fisiologia , Epitopos de Linfócito B/fisiologia , Humanos , Linfoma de Células B , Camundongos , Dados de Sequência Molecular , NF-kappa B/fisiologia , Subunidade p50 de NF-kappa B , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-rel , Relação Estrutura-Atividade , Fator de Transcrição RelB , Transfecção , Células Tumorais CultivadasRESUMO
Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL-6 secretion, isotype switching, and up-regulation of surface molecules. TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to date, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytoplasmic tail of CD40, but previous studies have principally examined transient overexpression of TRAF6 in cells that do not normally express CD40. Thus, we examined the role of TRAF6 in CD40-mediated B lymphocyte effector functions using two approaches. We produced and stably expressed in mouse B cell lines a human CD40 molecule with two cytoplasmic domain point mutations (hCD40EEAA); this mutant fails to bind TRAF6, while showing normal association with TRAFs 2 and 3. We also inducibly expressed in B cells a transfected "dominant-negative" TRAF6 molecule which contains only the C-terminal TRAF-binding domain of TRAF6. Using both molecules, we found that TRAF6 association with CD40 is important for CD40-induced IL-6 and Ig secretion, and that TRAF6 mediates its effects on CD40-stimulated Ig secretion principally through its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up-regulation of other surface molecules. Interestingly, however, although we could show TRAF6-dependent CD40-mediated activation of NF-kappaB in 293 kidney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígenos CD40/fisiologia , Proteínas/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Células Produtoras de Anticorpos/imunologia , Células Produtoras de Anticorpos/metabolismo , Linfócitos B/enzimologia , Antígenos CD40/biossíntese , Antígenos CD40/genética , Linhagem Celular , Ativação Enzimática/imunologia , Vetores Genéticos/síntese química , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Humanos , Interleucina-6/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Biossíntese de Proteínas , Proteínas/genética , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/genética , Fator 6 Associado a Receptor de TNF , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Signals delivered to antigen-presenting cells through CD40 are critical for the activation of immune responses. Intracellular tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are key elements of the signal transduction pathways of many TNF receptor family members, including CD40. We show for the first time that engagement of CD40 in intact B cells induces the rapid translocation of TRAF2 from the cytoplasm to the plasma membrane. We found that CD40 engagement also results in its recruitment, together with TRAF2 and TRAF3, to membrane microdomains, regions of the plasma membrane enriched in signaling molecules such as the Src family kinases. Using a membrane-permeable chelator of zinc or a mutant TRAF2 molecule, we show that the putative zinc-binding domains of TRAFs contribute to their recruitment to microdomains and to the downstream activation of c-Jun N-terminal kinase. We suggest that the zinc RING and zinc finger domains of TRAFs are required for communication between CD40 and microdomain-associated signaling molecules and may serve a similar role in the signal transduction pathways of other TNF receptor family members.
Assuntos
Antígenos CD40/fisiologia , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/imunologia , Animais , Linfócitos B , Linhagem Celular , Membrana Celular/imunologia , Quelantes/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenodiaminas/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Microscopia Confocal , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Recombinantes/metabolismo , Fator 2 Associado a Receptor de TNF , Fator 3 Associado a Receptor de TNF , TransfecçãoRESUMO
B cells are induced to express CD95 upon interaction with T cells. This interaction renders the B cells sensitive to CD95-mediated apoptosis, but ligation of proviability surface receptors is able to inhibit apoptosis induction. MHC class II is a key molecule required for Ag presentation to Th cells, productive T cell-B cell interaction, and B cell activation. We demonstrate here for the first time that MHC class II ligation also confers a rapid resistance to CD95-induced apoptosis, an affect that does not require de novo protein synthesis. Signaling through class II molecules blocks the activation of caspase 8, but does not affect the association of CD95 and Fas-associated death domain-containing protein. MHC class II ligation thus blocks proximal signaling events in the CD95-mediated apoptotic pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Transdução de Sinais/imunologia , Receptor fas/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Linfócitos B/enzimologia , Linfócitos B/metabolismo , Proteínas de Transporte/metabolismo , Caspase 8 , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Ativação Enzimática/imunologia , Proteína de Domínio de Morte Associada a Fas , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Hidrólise , Camundongos , Camundongos Endogâmicos BALB C , Biossíntese de Proteínas , Proteínas/metabolismo , Proteínas/fisiologia , Células Tumorais Cultivadas , Receptor fas/biossíntese , Receptor fas/metabolismoRESUMO
Signals delivered to B cells through CD40 are critical to the development of humoral immune responses. In this study we characterize regions of the 62-amino acid cytoplasmic domain of human CD40 (hCD40) that are essential for signal transduction and examine the functional consequences of mutations in these regions. A panel of mutant hCD40 molecules was stably expressed in mouse B cell lines and tested for its ability to stimulate Ab secretion, homotypic adhesion, and increased surface expression of B7, Fas, CD23, LFA-1, and intracellular adhesion molecule-1. Our results indicate that CD40 contains at least two major signaling determinants in the cytoplasmic domain: one disrupted by a truncation of 22 amino acids, and a second disrupted by the removal of 10 additional amino acids. The second determinant includes threonine 234, a residue previously shown to be important in CD40 signal transduction. Our functional analysis of alanine and serine substitutions at position 234 indicates that phosphorylation of this residue may be important for full CD40 signaling activity.
Assuntos
Antígenos CD40/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Antígenos CD40/química , Antígenos CD40/genética , Adesão Celular , Agregação Celular , Linhagem Celular , Cricetinae , Citometria de Fluxo , Humanos , Imunoglobulina M/metabolismo , Molécula 1 de Adesão Intercelular , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores de IgE/metabolismo , Relação Estrutura-Atividade , Receptor fas/metabolismoRESUMO
The imidazoquinoline R-848, originally identified as a highly effective antiviral agent, has recently been shown to be capable of potent B lymphocyte activation. The B cell-activating properties of R-848 are strikingly similar to the effects of the CD40 ligand CD154. The present study demonstrates that this similarity extends to the intracellular signaling pathways triggered by the compound, although both overlapping and distinct mechanisms of signaling were seen. Like CD40 ligation, R-848 stimulated activation of the stress-activated protein kinases c-Jun kinase and p38 and activated the NF-kappaB family of transcription factors. Both R-848- and CD40-mediated B cell differentiation were dependent upon NF-kappaB activation, although the relative importance of individual NF-kappaB family members appeared to differ between R-848- and CD40-mediated signals. Both signals were partially dependent upon induction of TNF-alpha and IL-6, and the cytoplasmic adaptor molecule TNF receptor-associated factor 2 is involved in both R-848- and CD40-mediated differentiation.
Assuntos
Adjuvantes Imunológicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Imidazóis/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Animais , Células Produtoras de Anticorpos/efeitos dos fármacos , Células Produtoras de Anticorpos/imunologia , Células Produtoras de Anticorpos/metabolismo , Linfócitos B/enzimologia , Linfócitos B/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Interleucina-6/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/imunologia , Fator 2 Associado a Receptor de TNF , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Testing of pharmacological agents that affect growth of epidermal keratinocytes (EK) requires a standardized assay. We have developed an assay measuring net effects of stimulatory (e.g. growth factors), inhibitory (e.g. methotrexate) or toxic (e.g. Triton X-100) compounds. The amount of crystal violet staining viable EK attached to the wells of standard 96-well microplates is measured in situ using an ELISA plate reader. Optical density readings are directly converted into cell counts by computer software. Counts obtained by this method strongly correlate with the results obtained using the [3H]thymidine uptake assay and direct cell counts. The assay standardizes measurements of nonimmortalized EK lines with different innate proliferative properties and allows accurate quantitation of EK numbers in the range of 2,500-500,000 EK/well.