RESUMO
Signals generated through CD28-B7 and CD40 ligand (CD40L)-CD40 interactions have been shown to be crucial for the induction of long-term allograft survivability. We have recently demonstrated that humanized anti-CD40L (hu5C8) prevents rejection of mismatched renal allografts in primates. To investigate potential mechanisms of CD40L-induced allograft acceptance, we coimmobilized hu5C8 with suboptimal amounts of anti-CD3 to stimulate CD4(+) T cells. We now report that anti-CD3/CD40L costimulation results in CD28-independent activation and subsequent deletion of resting T cells. Coligation of CD3 and CD40L increased expression of CD69, CD25, and CD54 on CD4(+) T cells. We also found that costimulation with anti-CD3/CD40L resulted in enhanced production of interleukin (IL)-10, interferon gamma, and tumor necrosis factor alpha but not IL-2 or IL-6. Interestingly, after several days, anti-CD3/CD40L-mediated activation was followed by apoptosis in a significant population of cells. Consistent with that observation, anti-CD3/CD40L did not enhance the antiapoptotic proteins Bcl-2 and Bcl-xL. Further, the addition of CD28 at 24 h failed to rescue those cells induced to die after costimulation with anti-CD3/CD40L. Together, these data suggest that the graft-sparing effect of hu5C8 in vivo may result in part from early and direct effects on CD4(+) T cells, including a vigorous induction of immunomodulatory cytokines and/or apoptosis of allograft-specific T cells.
Assuntos
Apoptose , Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/fisiologia , Citocinas/biossíntese , Glicoproteínas de Membrana/fisiologia , Antígenos CD/análise , Antígenos CD/fisiologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Antígenos CD40/genética , Ligante de CD40 , Células Cultivadas , Citocinas/genética , Regulação da Expressão Gênica , Humanos , Imunoglobulina G/farmacologia , Interferon gama/biossíntese , Interferon gama/genética , Interleucinas/biossíntese , Interleucinas/genética , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , Glicoproteínas de Membrana/imunologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transfecção , Células Tumorais CultivadasRESUMO
In vitro evidence suggests that memory CD4(+) cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA+ CD4(+) (naïve) and CD45RO+ CD4(+) (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4(+) cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4(+) subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta upon stimulation. Neutralization of these beta-chemokines rendered memory CD4(+) cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and beta-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4(+) T cells serve as the targets for early rounds of HIV-1 replication.
Assuntos
Antígenos CD , Antígenos de Diferenciação/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Memória Imunológica , NAD+ Nucleosidase/imunologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Sequência de Bases , Complexo CD3/imunologia , Quimiocinas CC/imunologia , Primers do DNA , Infecções por HIV/transmissão , Humanos , Glicoproteínas de Membrana , Receptores de HIV/metabolismo , Replicação Viral/imunologiaRESUMO
We have assessed the functional effects of a panel of CTLA-4 mAbs on resting human CD4+ T cells. Our results demonstrate that some CTLA-4 mAbs can inhibit proliferative responses of resting CD4+ cells and cell cycle transition from G0 to G1. The inhibitory effects of CTLA-4 were evident within 4 h, at a time when cell surface CTLA-4 expression remained undetectable. Other CTLA-4 mAbs had no detectable inhibitory effects, indicating that binding of Ab to CTLA-4 alone is not sufficient to mediate down-regulation of T cell responses. Interestingly, while IL-2 production was shut off, inhibitory anti-CTLA-4 mAbs permitted induction and expression of the cell survival gene bcl-X(L). Consistent with this observation, cells remained viable and apoptosis was not detected after CTLA-4 ligation.
Assuntos
Antígenos de Diferenciação/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Imunoconjugados , Interleucina-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Abatacepte , Anticorpos Monoclonais , Antígenos CD , Antígeno CTLA-4 , Ciclo Celular , Células Cultivadas , Expressão Gênica , Humanos , Ativação Linfocitária , RNA Mensageiro/genética , Transdução de Sinais , Proteína bcl-XRESUMO
This study examined the role of transforming growth factor beta 1 (TGF-beta 1) in monocytic differentiation of hematopoietic cells. TGF-beta 1 and retinoic acid (RA) inhibited HL-60 cell growth in a dose-dependent fashion. Treatment of HL-60 cells with a combination of TGF-beta 1 and a 50% optimal dose of RA (RA + TGF-beta 1) resulted in increased growth suppression compared to the individual treatments. Morphological studies revealed that TGF-beta 1 induced promonocytic differentiation (68%), RA induced granulocytic differentiation (98%), and RA + TGF-beta 1 induced monocytic (54%) and granulocytic (46%) differentiation of HL-60 cells. Induction of the monocyte-specific marker, nonspecific esterase, was markedly increased by TGF-beta 1 and RA + TGF-beta 1 treatment but not by RA treatment. Both TGF-beta 1 treatment and RA treatment increased TGF-beta ligand and TGF-beta receptor protein and mRNA levels. To determine whether RA mediated HL-60 cell growth inhibition and differentiation through the autocrine expression of TGF-beta 1, experiments using TGF-beta 1 antisense oligonucleotides or TGF-beta 1-neutralizing antibodies were conducted. TGF-beta 1 antisense oligonucleotides and neutralizing antibodies partially blocked RA-induced inhibition of proliferation, and TGF-beta 1 antisense oligonucleotides reversed RA-induced granulocytic maturation, demonstrating that RA signals autocrine expression of TGF-beta 1 and TGF-beta receptors. The effect of TGF-beta 1 on normal hematopoiesis was also studied using primary human fetal liver cells. TGF-beta 1 alone and in the presence of interleukin 3 promoted macrophage differentiation of primitive fetal liver cells. Cell surface expression of the monocyte/macrophage-specific marker c-fms was increased 3.1-fold following TGF-beta 1 treatment. In addition, TGF-beta 1-treated cells displayed a 51% increase in phagocytosis as compared to interleukin 3-treated control cells. These studies define a role for TGF-beta 1 in the autocrine and paracrine regulation of monocyte/macrophage differentiation.
Assuntos
Hematopoese/fisiologia , Monócitos/citologia , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta/farmacologia , Tretinoína/farmacologia , Biomarcadores , Diferenciação Celular , Divisão Celular , Células Cultivadas , Esterases/análise , Expressão Gênica , Granulócitos/citologia , Células HL-60 , Humanos , Interleucina-3/farmacologia , Fígado/citologia , Fígado/embriologia , Oligonucleotídeos Antissenso , Fagocitose , Receptor de Fator Estimulador de Colônias de Macrófagos/análise , Receptores de Fatores de Crescimento Transformadores beta/análiseRESUMO
One- or two-gram doses of ceftriaxone were administered intravenously to 30 patients before cataract extraction. With the 1-g dose, mean aqueous humor concentrations of 0.93 and 0.88 microgram/mL were found at approximately 2 and 12 hours after administration, respectively. With the 2-g dose, a mean level of 2.47 micrograms/mL was observed at two hours; levels of more than 2 micrograms/mL were found in two patients 13 hours after administration. Both the 1- and 2-g doses thus produce aqueous humor levels many times higher than the minimum inhibitory concentration of ceftriaxone for 90% of most Enterobacteriaceae, excluding Pseudomonas. Concentrations adequate for Staphylococcus aureus and Staphylococcus epidermidis were not, however, obtained.