RESUMO
One challenge in caring for patients with systemic lupus erythematosus (SLE) is a paucity of approved therapeutics for treatment of the diverse disease manifestations. In the last 60 years, only one drug, belimumab, has been approved for SLE treatment. Critical evaluation of investigator initiated and pharma-sponsored randomized controlled trials (RCTs) highlights barriers to successful drug development in SLE, including disease heterogeneity, inadequate trial size or duration, insufficient dose finding before initiation of large trials, handling of background medications, and choice of primary endpoint. Herein we examine lessons learned from landmark SLE RCTs and subsequent advances in trial design, as well as discuss efforts to address limitations in current SLE outcome measures that will improve detection of true therapeutic responses in future RCTs.
Assuntos
Imunossupressores/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Ensaios Clínicos Fase III como Assunto/métodos , Aprovação de Drogas , Indústria Farmacêutica , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto/normas , Resultado do Tratamento , Estados Unidos , United States Food and Drug AdministrationRESUMO
Lupus patients are in need of modern drugs to treat specific manifestations of their disease effectively and safely. In the past half century, only one new treatment has been approved by the US Food and Drug Administration (FDA) for systemic lupus erythematosus (SLE). In 2014-2015, the FDA approved 71 new drugs, only one of which targeted a rheumatic disease and none of which was approved for use in SLE. Repositioning/repurposing drugs approved for other diseases using multiple approaches is one possible means to find new treatment options for lupus patients. "Big Data" analysis approaches this challenge from an unbiased standpoint whereas literature mining and crowd sourcing for candidates assessed by the CoLTs (Combined Lupus Treatment Scoring) system provide a hypothesis-based approach to rank potential therapeutic candidates for possible clinical application. Both approaches mitigate risk since the candidates assessed have largely been extensively tested in clinical trials for other indications. The usefulness of a multi-pronged approach to drug repositioning in lupus is highlighted by orthogonal confirmation of hypothesis-based drug repositioning predictions by "Big Data" analysis of differentially expressed genes from lupus patient samples. The goal is to identify novel therapies that have the potential to affect disease processes specifically. Involvement of SLE patients and the scientists that study this disease in thinking about new drugs that may be effective in lupus though crowd-sourcing sites such as LRxL-STAT (www.linkedin.com/in/lrxlstat) is important in stimulating the momentum needed to test these novel drug targets for efficacy in lupus rapidly in small, proof-of-concept trials conducted by LuCIN, the Lupus Clinical Investigators Network (www.linkedin.com/in/lucinstat).
Assuntos
Biologia Computacional/métodos , Reposicionamento de Medicamentos/métodos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Animais , Crowdsourcing , Mineração de Dados , Genômica , Humanos , Lúpus Eritematoso Sistêmico/genéticaRESUMO
The mechanism of toxicity for cytolytic lymphocytes of Leu-Leu-OMe and related dipeptide derivatives was examined. Selective inhibition of dipeptidyl peptidase I (DPPI), a lysosomal thiol protease highly enriched in cytotoxic lymphocytes, prevented all natural killer (NK) toxic effects of such agents. However, many DPPI substrates were found to possess no NK toxic properties. For some such agents, this lack of NK toxicity appeared to be related to the lack of uptake by lymphocytes. In this regard, Leu-Leu-OMe was found to be incorporated by lymphocytes and monocytes via a saturable facilitated transport mechanism with characteristics distinct from previously characterized mammalian dipeptide transport processes. This novel transport process was found to be specific for dipeptides composed of selective L-stereoisomer amino acids and enhanced by hydrophobic ester or amide additions to the COOH terminus of dipeptides. Maximal rates of Leu-Leu-OMe uptake by T8 and NK cell-enriched peripheral blood lymphocytes (PBL) were four- to sixfold higher than for T4-enriched PBL or PBL depleted of Leu-Leu-OMe-sensitive cytotoxic lymphocytes. All dipeptide amides or esters with NK toxic properties were found to act as competitive inhibitors of [3H]Leu-Leu-OMe uptake by PBL. However, some NK nontoxic DPPI substrates were found to be comparable with Leu-Leu-OMe in avidity for this transport process. Such agents were noted to possess one or more hydrophilic amino acid side chains and were found not to mediate red blood cell lysis when subjected to the acyl transferase activity of DPPI. Thus, uptake by a dipeptide-specific facilitated transport mechanism and conversion by DPPI to hydrophobic polymerization products with membranolytic properties were found to be common features of NK toxic dipeptide derivatives. The presence of a previously unreported dipeptide transport mechanism within blood leukocytes and the selective enrichment of the granule enzyme, DPPI, within cytotoxic effector cells of lymphoid or myeloid lineage appear to afford a unique mechanism for the targeting of immunotherapeutic reagents composed of simple dipeptide esters or amides.
Assuntos
Dipeptídeos/metabolismo , Dipeptídeos/toxicidade , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacos , Aciltransferases/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Transporte Biológico Ativo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Catepsina C , Dipeptídeos/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Eritrócitos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Dados de Sequência Molecular , Linfócitos T Citotóxicos/metabolismo , TemperaturaRESUMO
The nature of the physical interaction between guinea pig non-glass-adherent lymphoid cells and syngeneic macrophages in vitro was investigated. This cellular interaction was found to require the presence of metabolically intact macrophages but neither serum nor antigen. Peritoneal, splenic, or alveolar macrophages were significantly more capable of interacting with thymocytes than either polymorphonuclear leukocytes or fibroblasts. The role of the non-glass-adherent cell was passive in that heat-killed or metabolically poisoned thymocytes were bound by normal macrophages. Two normal lymphoid cell populations, thymocytes, and lymph node lymphocytes, were bound to macrophages in significantly larger numbers than either L(2)C leukemia cells or erythrocytes. Thus, specificity for each of the participant cell types was demonstrated. These data indicate that macrophages possess a unique ability to recognize and bind lymphocytes and thymocytes by a mechanism which is distinguishable from other known macrophage receptors.
Assuntos
Linfócitos/fisiologia , Macrófagos/fisiologia , Timo/citologia , Animais , Antígenos , Sítios de Ligação , Contagem de Células , Técnicas de Cultura , Cobaias , Temperatura Alta , Contagem de Leucócitos , Linfócitos/imunologia , Linfócitos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Microscopia Eletrônica , Neuraminidase/farmacologia , Timo/imunologia , Tripsina/farmacologiaRESUMO
The effect of specific antigen on the development of physical interactions between lymph node lymphocytes (LNL) obtained from animals which had been immunized to that antigen and macrophages was examined. We found that the presence of antigen, either limited to the macrophage () or free in the medium, profoundly increased the degree of ) or free in the medium, profoundly increased the degree of Mphi-LNL interaction observed. This enhanced interaction was dependent on the coincidence in the cultures of Mphi bearing antigen and LNL from animals specifically immunized to that antigen. Although antigen-independent interactions developed equally well between syngeneic and allogeneic combinations of lymphocytes and macrophages, antigen mediated interactions required that macrophages and lymphocytes be syngeneic. Prolongation of antigen-mediated Mphi-LNL interactions resulted in the induction of LNL DNA synthesis, initially involving those lymphocytes physically associated with antigen-bearing Mphi. These studies are interpreted to indicate that physical interaction between immune lymphocytes and antigen-bearing Mphi represents a morphological correlate of the functional activation of immune lymphocytes. Further, it is suggested that the physical events involved in lymphocyte proliferation may proceed sequentially from antigen-independent reversible binding of lymphocytes by macrophages to prolonged antigen-stabilized interaction eventuating in the triggering of specifically immune lymphocytes.
Assuntos
Reações Antígeno-Anticorpo , Linfócitos/imunologia , Macrófagos/imunologia , Animais , Antígenos , Autorradiografia , Sítios de Ligação de Anticorpos , Células Cultivadas , DNA/biossíntese , Epitopos , Cobaias , Antígenos de Histocompatibilidade , Linfonodos/imunologia , Linfócitos/metabolismo , Timidina/metabolismo , TrítioRESUMO
The leukocyte function-associated antigen 1 (LFA-1) molecule is well established as a surface protein involved in cellular adhesion and interaction, but there has been little information about whether engagement of this molecule can also directly modify cellular activation. These studies demonstrate that crosslinking the LFA-1 molecule on human T cell clones transmits a unique signal to the cell. Crosslinking LFA-1 alone did not increase intracellular calcium ([ CA2+]i), nor did crosslinking LFA-1 activate the cells as measured by IL-2 production or [3H]thymidine incorporation. However, when CD3 and LFA-1 were crosslinked, a more prolonged calcium signal was observed than when CD3 alone was crosslinked. Moreover, IL-2 production and DNA synthesis were greatly augmented. These responses could be demonstrated when LFA-1 was crosslinked via either the alpha or the beta chain, and required surface expression of the LFA-1 molecule as no enhancement was observed in T cell clones from a child with leukocyte adhesion deficiency. The enhancement of cellular activation by LFA-1 did not require that it be directly crosslinked to the CD3 complex. Thus, crosslinking LFA-1 alone with isotype-specific secondary antibodies on cells also pretreated with an anti-CD3 mAb of a different Ig isotype stimulated the cells as effectively as crosslinking both surface antigens with GaMIg. Similarly, a delayed, but sustained increase in [Ca2+]i was elicited. This increase in [Ca2+]i and the enhanced functional responses required engagement of CD3 with an intact bivalent anti-CD3 mAb, as crosslinking LFA-1 on cells also reacted with Fab fragments of an anti-CD3 mAb did not increase [Ca2+]i, nor activate the cells. These data indicate that LFA-1 can convey activation signals to T cells. Synergism in signaling can be observed upon crosslinking of LFA-1 and independently crosslinking CD3. In the physiologic interaction between T cells and accessory cells, the interaction of LFA-1 with its ligand, intercellular adhesion molecule 1, may therefore not only facilitate cellular adhesion, but also may amplify T cell activation by delivering costimulatory signals.
Assuntos
Antígenos de Diferenciação/fisiologia , Ativação Linfocitária , Linfócitos T/imunologia , Anticorpos Monoclonais , Reações Antígeno-Anticorpo , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Antígenos de Superfície/fisiologia , Antígenos CD11 , Antígenos CD18 , Complexo CD3 , Cálcio/metabolismo , Moléculas de Adesão Celular , Células Cultivadas , Antígenos HLA/imunologia , Humanos , Técnicas In Vitro , Interleucina-2/biossíntese , Antígeno-1 Associado à Função Linfocitária , Substâncias Macromoleculares , Glicoproteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Fatores de TempoRESUMO
The structural requirements for signal transduction by class I major histocompatibility complex (MHC) molecules were examined. Native or mutant HLA-A2 or HLA-B27 constructs lacking most of their cytoplasmic domains were co-transfected with pSV2neo into Jurkat cells. Transfection of either native or mutant constructs resulted in a comparable expression of the gene products. Stimulation of transfectants expressing either native or truncated A2 or B27 molecules with specific mAb evoked an increase in [Ca2+]i upon crosslinking. Moreover, crosslinking native or truncated A2 or B27 induced IL-2 production upon co-stimulation with phorbol myristate acetate. These results confirm that crosslinking class I MHC molecules transduces an activation signal to human T cells. Effective signaling was observed when all but four of the intracytoplasmic residues were deleted, indicating that signal transduction does not require this portion of the molecule.
Assuntos
Antígeno HLA-A2/fisiologia , Antígeno HLA-B27/fisiologia , Transdução de Sinais , Linfócitos T/imunologia , Animais , Cálcio/análise , Citoplasma/fisiologia , Antígeno HLA-A2/genética , Antígeno HLA-B27/genética , Interleucina-2/biossíntese , Camundongos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The dynamics of immunoglobulin rearrangements and selection, which depend on age, antigen exposure and tolerance functions, are only partly understood. Thus, we analyzed and compared the lambda chain immunoglobulin repertoire of individual IgD+ human neonatal B cells with the adult peripheral B cell VlambdaJlambda repertoire. Some Vlambda genes, 4C, 2A2, 2B2, 5A, 1G and 4B, were overexpressed in the non-productive neonatal repertoire, whereas other Vlambda genes (2E, 2A2, 3H, 2B2, 1C and 1G) were overexpressed in the productive repertoire. The adult B cell repertoire revealed nearly the same predominance of genes in the non-productive and productive repertoire. A comparison of the non-productive and productive repertoire indicated that the genes 3H and 1C were positively selected, whereas the genes 4C, 2A1, 3I, 5A, 9A, 4A and 4B were negatively selected. All four functional Jlambda genes were used in both repertoires. Jlambda2/3 was used mainly. Insertions of non-templated nucleotides at the VlambdaJlambda-junction by the enzyme TdT were less frequent as compared to the adult, but the CDR3 length was the same. Comparison of CD5+IgD+ and CD5-IgD+ B cells revealed no differences between neonatal productive rearrangements. However, the genes 1C and 1G were used more often in the non-productive repertoire of CD5+ B cells, whereas gene 4B was used significantly more frequent in CD5- B cells. These data provide evidence that the primary usage and subsequent selection of Vlambda genes in the neonate are surprisingly comparable with the adult. This suggests that selection into the productive Vlambda repertoire in principal might be driven mainly by autoantigens in the newborn, as well as in adulthood, since newborns have not been exposed to exogenous antigens.
Assuntos
Linfócitos B/imunologia , Genes de Imunoglobulinas , Cadeias lambda de Imunoglobulina/genética , Linfócitos B/enzimologia , Antígenos CD5/imunologia , Regiões Determinantes de Complementaridade/genética , Análise Mutacional de DNA , DNA Nucleotidilexotransferase/metabolismo , Exonucleases/metabolismo , Sangue Fetal/citologia , Rearranjo Gênico de Cadeia Leve de Linfócito B , Humanos , Imunoglobulina D/genética , Cadeias J de Imunoglobulina/genética , Recém-NascidoRESUMO
Interleukin 21 (IL21) belongs to a family of cytokines that bind to a composite receptor consisting of a private receptor (IL21R) and the common cytokine receptor gamma chain (gamma(C)). The IL21R is widely distributed on lympho-haematopoietic cells and IL21 impacts a number of cell types, including CD8+ memory T cells, NK cells and subsets of CD4 memory T cells. One essential role of IL21 is the promotion of B-cell activation and differentiation or death during humoral immune responses. Increased IL21 production is characteristic of certain autoimmune diseases and is likely to contribute to autoantibody production as well as pathological features of autoimmune disease. The critical role of IL21 in promoting humoral immune responses makes it an important focus of potential therapeutic interventions in conditions characterised by overproduction of pathogenic autoantibodies.
Assuntos
Doenças Autoimunes/imunologia , Interleucinas/imunologia , Animais , Artrite Reumatoide/imunologia , Doenças Autoimunes/terapia , Linfócitos B/imunologia , Humanos , Switching de Imunoglobulina/imunologia , Interleucinas/antagonistas & inibidores , Interleucinas/genética , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/imunologia , CamundongosRESUMO
OBJECTIVE: To investigate the expressions of Foxp3 and CD25 on CD4(+) T cells from patients with new-onset systemic lupus erythematosus (SLE) and assess their clinical significance. METHODS: 10 patients with active (systemic lupus erythematosus Disease Activity Index (SLEDAI) >or=10) and 11 with inactive (SLEDAI
Assuntos
Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/sangue , Lúpus Eritematoso Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Tolerância Imunológica , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-2/sangue , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Linfócitos T Reguladores/imunologiaRESUMO
It has been suggested that D-penicillamine is active in rheumatoid arthritis because of its capacity to function as a selective inhibitor of T lymphocyte function. The basis for the immunosuppressive action of this drug as well as mechanisms whereby the effect of D-penicillamine could be modified by elements of rheumatoid synovial tissue were examined. As previously reported, D-penicillamine, in the presence of copper ions markedly inhibited mitogen-induced human T lymphocyte DNA synthesis. Since the vast majority of copper in the body exists as an integral part of the ceruloplasmin molecule, the capacity of this cuproprotein to augment D-penicillamine-mediated inhibition of T cell function was examined. The requirement for copper ions could be entirely replaced by purified ceruloplasmin, which had been depleted of nonspecifically bound copper by passage over Chelex-100 columns. The mechanism by which D-penicillamine in the presence of either copper ions or ceruloplasmin caused inhibition of T lymphocyte responsiveness was examined. Partial protection from this inhibitory effect was accomplished by sodium borohydride. While superoxide dismutase had no protective effect, catalase was found to protect lymphocyte responsiveness totally from the inhibitory action of D-penicillamine and either copper ions or ceruloplasmin. Similarly, horseradish peroxidase and myeloperoxidase also protected responsiveness from these inhibitors while boiled catalase was without effect. These results indicate that inhibition of T lymphocyte responsiveness resulted from the generation of hydrogen peroxide. Since a number of cells likely to be present at chronic inflammatory sites, such as mononuclear phagocytes, contain enzymatic mechanisms to degrade hydrogen peroxide, the modulatory influence of these cells on the inhibition of T cell function caused by D-penicillamine and copper was examined. Monocytes, whose function was not suppressed by D-penicillamine and copper, were found to protect T lymphocyte responsiveness from the inhibitory effects of either the mixture of D-penicillamine and CuSO4 or of hydrogen peroxide. By contrast, endothelial cells, fibroblasts, or cells obtained from enzyme-digested noninflamed synovium could not protect T cells from the inhibitory effects of D-penicillamine and copper. Protection of T cells was afforded by means of a heat labile, azide-sensitive soluble factor present in lysates of human monocytes. These results indicate that the mechanism whereby D-penicillamine in the presence of copper or ceruloplasmin inhibits T lymphocyte responsiveness involves the generation of hydrogen peroxide and that other neighboring cells likely to be found w
Assuntos
Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Penicilamina/farmacologia , Linfócitos T/imunologia , Adulto , Azidas/farmacologia , Catalase/metabolismo , Sobrevivência Celular , Ceruloplasmina/farmacologia , Cobre/metabolismo , Cobre/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Imunidade Celular , Monócitos/enzimologia , Monócitos/imunologia , Monócitos/fisiologia , Azida Sódica , Linfócitos T/efeitos dos fármacosRESUMO
The effect of d-penicillamine (Pen) and mixtures of Pen and copper sulfate on the capacity of normal human peripheral blood mononuclear cells (PBM) to generate immunoglobulin-secreting cells (ISC) in response to the T-cell-dependent polyclonal B-cell activators pokeweed mitogen (PWM) and staphylococcal protein A (SPA) was examined. PBM obtained from normal individuals were incubated for 1-2 h at 37 degrees C with medium alone, Pen, CuSO(4), or a mixture of Pen and CuSO(4). After washing, the cells were incubated for 6-7 d with PWM or SPA and then, with a reverse hemolytic plaque assay, assayed for the number of ISC generated. Preincubation of PBM with either Pen (100 mug/ml) or CuSO(4) (2 mug/ml) did not alter the subsequent capacity of the cells to generate ISC in response to PWM or SPA. In contrast, responsiveness to both mitogens was nearly abolished when PBM were similarly preincubated with a mixture of Pen and CuSO(4). Inhibition of responsiveness could not be ascribed to cell death, carry-over of the inhibitors, or an alteration in the concentration of PWM or the length of incubation yielding maximum responses. Co-culture experiments demonstrated that Pen and CuSO(4) preincubation had not caused augmented suppressor cell function. Experiments in which PBM were separated into adherent and nonadherent populations indicated that Pen and CuSO(4) preincubation inhibited the responsiveness of the nonadherent cells but did not alter the accessory cell function of monocytes. To determine whether Pen and CuSO(4) preincubation effected T- or B-cell function, PBM were separated into B- and T-cell-enriched populations, individually preincubated with Pen and CuSO(4), and then co-cultured with PWM. The results indicated that Pen and CuSO(4) markedly inhibited helper T-cell function and had little effect on the capacity of B cells to generate ISC. The observation that in the presence of CuSO(4) Pen inhibits helper T-cell activity may, in part, explain the therapeutic efficacy of Pen in rheumatoid arthritis and especially the capacity of Pen therapy to decrease antiglobulin titers in treated patients.
Assuntos
Cobre/farmacologia , Penicilamina/farmacologia , Linfócitos T/efeitos dos fármacos , Adulto , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Humanos , Imunoglobulinas/metabolismo , Técnicas In Vitro , Mitógenos de Phytolacca americana/farmacologia , Sulfatos/farmacologia , Linfócitos T/imunologiaRESUMO
The effect of soluble or immobilized MAb directed at various additional surface proteins on the proliferation of highly purified T4 cells induced by two immobilized MAb to CD3, OKT3 and 64.1, was examined. High density 64.1 stimulated nearly all T4 cells to enter and progress through the cell cycle. Maximal T4 cell proliferation required stimulation with immobilized 64.1 throughout the length of the incubation and was not effected by any of the additional soluble or immobilized MAb employed. In contrast, low density immobilized 64.1 and all densities of immobilized OKT3 employed stimulated a minority of the cells to enter the cell cycle and proliferate. Immobilized MAb directed at CD2, class I major histocompatibility complex (MHC) encoded gene products or CD11a (LFA-1) dramatically enhanced the response, whereas soluble MAb directed at these determinants did not. Both immobilized and soluble MAb directed at CD5 and CD28 (Tp44) enhanced responses, but they were less effective than immobilized MAb to CD2, LFA-1 or HLA-A,B,C. Soluble anti-CD4 MAb inhibited responses somewhat, whereas immobilized anti-CD4 enhanced responses. Costimulation was observed when MAb to CD3 and class I MHC molecules but not CD2, LFA-1 or CD4 were immobilized to separate surfaces. The data suggest that when anti-CD3 stimulation is suboptimal, responses can be enhanced by MAb to CD5 or CD28 (Tp44) or by immobilized MAb to CD4, CD2, CD11a (LFA-1), or class I MHC encoded gene products. Although crosslinking of CD4, CD2, or CD11a with CD3 may be necessary for costimulation, immobilized MAb to CD3 and class I MHC molecules appear to deliver independent signals that result in enhanced T4 cell activation and proliferation.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Antígenos de Superfície/imunologia , Complexo CD3 , Epitopos/imunologia , HumanosRESUMO
In contrast to adult lymphocytes, neonatal lymphocytes secrete minimal amounts of Ig in response to stimulation with immobilized MAb to CD3. This deficiency could be overcome by the addition of supplemental IL-2, IL-4, or IL-6, resulting in the secretion of all Ig isotypes. There were no major differences in the distribution of Ig isotypes secreted in response to the cytokines alone or in combination. The Ig secreted in response to IL-4 or IL-6 was inhibited by MAb to CD25, suggesting that the effects of IL-4 and IL-6 were dependent on IL-2. Stimulation of neonatal lymphocytes with anti-CD3 was sufficient to induce expression of IL-2 receptors (CD25) on both T and B cells. IL-4 exerted direct effects on neonatal T cells by increasing IL-2 production and promoting IL-6 production by anti-CD3-stimulated neonatal lymphocytes. Antibody to IL-4 or IL-6 did not inhibit Ig secretion in response to IL-2 and antibody to IL-6 did not consistently inhibit Ig secretion in response to IL-4. Finally, in the presence of cyclosporin, anti-CD3-stimulated neonatal lymphocytes secreted Ig only with the combination of IL-2 and IL-4. These results have delineated unique, but not Ig isotype-specific, effects of cytokines in supporting Ig secretion by anti-CD3-stimulated neonatal lymphocytes. Deficient production of these cytokines is likely to contribute to the decreased capacity of neonatal lymphocytes to generate an Ig response.
Assuntos
Citocinas/farmacologia , Sangue Fetal/imunologia , Imunoglobulinas/metabolismo , Linfócitos/imunologia , Adulto , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/imunologia , Complexo CD3 , Ciclosporinas/farmacologia , Antígenos de Histocompatibilidade/análise , Humanos , Recém-Nascido , Interferon gama/farmacologia , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Interleucina-6/biossíntese , Interleucina-6/farmacologia , Antígenos Comuns de Leucócito , Linfócitos/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Gold sodium thiomalate (GST) inhibited in vitro antigen- and mitogen-triggered human lymphocyte DNA synthesis. Inhibition of responsiveness was observed with concentrations of GST equivalent to gold levels found in serum or tissues of patients receiving chrysotherapy, Inhibition was dependent upon the gold ion itself since GST and gold chloride were both inhibitory whereas thiomalic acid was not. Inhibition could not be explained by nonspecific killing of cells or by an alteration in the kinetics of the responses. GST inhibited mitogen-induced proliferation most effectively when present from the initiation of culture and could not inhibit the responsiveness of cells which previously had been activated by concanvalin A. These findings indicated that GST blocked a critical early step in lymphocyte activation. The degree of GST-induced inhibition of proliferation was increased in cultures of cells partially depleted of monocytes. Moreover, inhibition was reversed by supplementation of these cultures with purified monocytes. These observations suggested that GST blocked thymus-derived (T)-lymphocyte activation by interfering with a requisite function of the monocyte population in initiating such responses. Prolonged incubation of peripheral blood mononuclear cells with GST resulted in diminished mitogen responsiveness upon subsequent culture in the absence of gold. The addition of fresh monocytes restored responsiveness to these populations. Furthermore, preincubation of purified monocytes with GST rendered them deficient in their ability to support mitogen-induced T-lymphocyte proliferation on subsequent culture. These observations indicate that the major effect of GST results from interference with the functional capability of the monocyte population.
Assuntos
Ouro/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Antígenos , Concanavalina A/farmacologia , DNA/biossíntese , Humanos , Técnicas In Vitro , Lectinas/farmacologia , Linfócitos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Tiomalatos/farmacologia , Fatores de TempoRESUMO
Human low density lipoprotein (LDL, d = 1.020-1.050 g/ml) inhibits mitogen-stimulated T lymphocyte DNA synthesis. Because both LDL and transferrin bind to specific cell surface receptors and enter cells by the similar means of receptor-mediated endocytosis, and because transferrin is necessary for lymphocyte DNA synthesis, we investigated the possibility that LDL may inhibit mitogen-stimulated lymphocyte responses by interfering with transferrin metabolism. LDL inhibited mitogen-stimulated lymphocyte [3H]thymidine incorporation in a concentration-dependent manner. The degree of inhibition was most marked in serum-free cultures, but was also observed in serum-containing cultures. The addition of transferrin not only augmented mitogen-induced lymphocyte [3H]thymidine incorporation in serum-free medium but also completely reversed the inhibitory effect of LDL in both serum-free and serum-containing media. Similar results were obtained when lymphocyte proliferation was assayed by counting the number of cells in culture. Transferrin also reversed the inhibition of lymphocyte responses caused by very low density lipoproteins and by cholesterol. The ability of transferrin to reverse the inhibitory effect of lipoproteins was specific, in that native but not denatured transferrin was effective whereas a variety of other proteins were ineffective. These results indicate that LDL inhibits mitogen-stimulated lymphocyte responses by interfering with transferrin metabolism. LDL only inhibited lymphocyte responses after a 48-h incubation if present from the initiation of the culture. By contrast, transferrin reversed inhibition when added after 24 h of the 48-h incubation. LDL did not inhibit lymphocyte responses by nonspecifically associating with transferrin. In addition, the acquisition of specific lymphocyte transferrin receptors was not blocked by LDL. Moreover, transferrin did not prevent the binding and uptake of fluorescent-labeled LDL by activated lymphocytes. Furthermore, LDL did not prevent the binding of transferrin to its receptor. Finally, LDL inhibition did not require specific high affinity cell surface receptors for cholesterol transport by LDL because similar inhibition and reversal by transferrin were observed with lymphocytes from a patient with homozygous familial hypercholesterolemia. Thus, LDL alters lymphocyte responses in a non-LDL receptor-mediated way by interfering with transferrin metabolism after specific binding of transferrin to receptors on activated lymphocytes.
Assuntos
Imunossupressores/farmacologia , Lipoproteínas LDL/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/imunologia , Transferrina/metabolismo , Adulto , Colesterol/farmacologia , Humanos , Cinética , Lipoproteínas/farmacologia , Lipoproteínas IDL , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/farmacologia , Fito-Hemaglutininas/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/fisiologia , Receptores de LDL , Receptores da Transferrina , Linfócitos T/metabolismo , Transferrina/farmacologiaRESUMO
The role of prostaglandin E2 (PGE2) in the generation of immunoglobulin-secreting cells (ISC) from human peripheral blood B cells was examined. Initial studies demonstrated that monocyte (M phi)-mediated suppression of the generation of ISC in Staphylococcus aureus (SA)-stimulated cultures was mitigated by indomethacin, and thus suggested that the cyclooxygenase pathway products of arachidonic acid played a role in the regulation of B cell activation. The possibility that PGE2, one of the major products of this pathway generated by M phi-affected human B cell responses, was therefore investigated. PGE2 was found to cause concentration-dependent inhibition of the generation of ISC in pokeweed mitogen- or SA-stimulated B cell cultures supported by T cells. Studies were therefore carried out to determine whether PGE2 inhibited the production of necessary T cell factors or directly altered B cell responsiveness. Initially, the effect of PGE2 on the capacity of mitogen-stimulated cells to secrete a factor that supported the differentiation of B cells into ISC was investigated. Excessive numbers of M phi or PGE2 inhibited the production of B cell differentiation factor from mitogen-stimulated T cells. The effect of PGE2 on the capacity of B cells to differentiate into ISC was more complex. PGE2 inhibited the generation of ISC when B cells were stimulated with SA and B cell differentiation factor-containing T cell supernatants. PGE2-mediated inhibition of ISC generation was observed even when addition of PGE2 was delayed until after ISC first were detected in culture. By contrast, PGE2 caused only minimal inhibition of the generation of ISC cultures stimulated by T cell supernatants alone or protein A-free SA and T cell supernatants. These results suggested that SA-responsive B cells were particularly sensitive to inhibition by PGE2. Additional experiments supported the conclusion that B cell sensitivity to inhibition by PGE2 is augmented by the immunoglobulin cross-linking effects of protein A-containing SA. Overall, the results support the conclusion that PGE2 at physiologically relevant concentrations can influence human antibody responses by means of a direct inhibitory action on the responding B cell or an indirect one on the production of necessary T cell factors.
Assuntos
Linfócitos B/efeitos dos fármacos , Imunoglobulinas/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Prostaglandinas E/farmacologia , Dinoprostona , Substâncias de Crescimento/biossíntese , Humanos , Indometacina/farmacologia , Interleucina-4 , Linfocinas/biossíntese , Fagócitos/imunologia , Proteína Estafilocócica A/farmacologia , Staphylococcus aureus , Linfócitos T/imunologiaRESUMO
Neonatal T cell-B cell collaboration was investigated utilizing a system of T cell-dependent polyclonal B cell activation and Ig secretion. In this system, T cells activated by immobilized anti-CD3 provide a potent stimulus for Ig production by adult lymphocytes. By contrast, anti-CD3 stimulation of cord blood lymphocytes generated minimal numbers of Ig-secreting cells. Ig production by neonatal lymphocytes was enhanced by the addition of Staphylococcus aureus or secreted factors from mitogen-stimulated adult T cells. Supplementation with IL-2 resulted in the production of large amounts of IgM and small amounts of IgG and IgA, with less Ig produced than by comparable cultures of adult lymphocytes. Neonatal T cells proliferated and produced IL-2 in response to immobilized anti-CD3, and supported B cell proliferation and Ig secretion by adult B cells, although not as effectively as adult T cells. Supernatants from activated neonatal T cells were markedly limited in their capacity to support Ig production by adult B cells. Neonatal B cells could be induced to differentiate in response to anti-CD3-stimulated adult T cells. However, the amounts of IgG and IgA secreted were small compared to adult levels. These studies indicate a relative, but not absolute, functional deficiency of both neonatal B and T cells.
Assuntos
Linfócitos B/fisiologia , Linfócitos T/fisiologia , Adulto , Formação de Anticorpos , Antígenos de Diferenciação de Linfócitos T/imunologia , Complexo CD3 , Separação Celular , DNA/biossíntese , Sangue Fetal , Humanos , Imunoglobulinas/biossíntese , Recém-Nascido , Interleucina-2/biossíntese , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Mitogen-stimulated human T cell activation is absolutely dependent on the participation of a nonresponding accessory cell. In populations of human peripheral blood mononuclear cells, monocytes function as the requisite accessory cells. The possibility that cultured endothelial cells (EC) might also function as accessory cells was studied by examining the potential of endothelial cells to restore mitogen responsiveness to monocyte-depleted human T cells. Highly purified T cells were prepared by isolating cells rosetting with sheep erythrocytes and removing monocyte contamination by glass adherence and nylon wool column passage. When cultured at low cell density, T cells failed to respond to stimulation with various mitogenic lectins, whereas co-culture with monocytes restored responsiveness. Similarly, EC obtained from umbilical vein, pulmonary artery, and ovarian vein restored the capacity of T cells to respond to mitogens. Mitogen-stimulated T cell activation required viable endothelial cells. Moreover, effective endothelial T cell cooperation appeared to involve the establishment of cell-to-cell contact between EC and responding T cells. Accessory cell function was not a nonspecific property of all tissue culture cells as evidenced by the finding that human foreskin fibroblasts, lung fibroblasts, and HeLa cells were unable to restore responsiveness to monocyte-depleted T cells. These observations indicate that endothelial cells can support the induction of mitogen-induced T cell activation and suggest that cells lining blood vessels may play an active role in the initiation of immune responses in vivo.
Assuntos
Endotélio/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Células Cultivadas , Células HeLa/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/imunologia , Nitrosoguanidinas/farmacologia , Fito-Hemaglutininas/farmacologiaRESUMO
Polymerase chain reaction (PCR) technology was employed to examine peripheral blood and synovial T cells in patients with rheumatoid arthritis (RA) for biased utilization of T cell receptor (TCR) variable region (V) genes. Oligonucleotide primers specific for individual TCR V beta gene families were used to amplify TCR gene products in a semiquantitative assay of their relative utilization in unselected T cell populations. Mean V beta expression in 24 RA peripheral blood samples was very similar to that in a panel of 15 normal subjects, except for a slight decrease in V beta 13.2 expression. V beta utilization in 8 RA synovial tissue samples and 13 synovial fluid samples was compared to simultaneously obtained blood samples. Although heterogeneous patterns of skewed V beta utilization were observed, several significant trends emerged. By a number of approaches to data analysis, a statistically significant increase in expression of V beta 6 and V beta 15 in synovial T cells was documented. In addition, increased synovial expression of V beta 14 was found, but only in the synovial fluid samples. Reduced expression of V beta 1, V beta 4, V beta 5.1, V beta 10, V beta 16, and V beta 19 was also observed in synovial T cells. These results indicate that biased V beta gene utilization in different peripheral compartments of RA patients can be observed in unselected T cell populations, and are consistent with the conclusion that populations of T cells expressing these V beta gene products may be involved in the pathogenesis of the disease.