RESUMEN
Chemokines play critical roles in leukocyte recruitment into sites of inflammation such as rheumatoid arthritis (RA). While chemokines immobilized on endothelium (solid-phase), but not soluble chemokines, direct rolling leukocytes to firmly adhere to endothelium, soluble and solid-phase chemokine gradients may play important roles in leukocyte extravasation into the tissue. In this study, we have sought to determine (1) if chemokines can be immobilized on structures in the extravascular space, (2) the mechanisms by which chemokines may be immobilized, and (3) if different chemokines have similar potentials to form solid-phase gradients. While secreted alkaline phosphatase (SEAP)-tagged chemokines SLC (CCL21), TARC (CCL17), and RANTES (CCL5) bound to mast cells and the extracellular matrix (ECM) in RA synovium under physiologic salt conditions, MCP1 (CCL2), MIP1 alpha (CCL3), MIP1 beta (CCL4), and fractalkine (FKN, CX3CL1) fusion proteins did not detectably bind. Chemokine binding to ECM and mast cells in situ and to immobilized heparin was inhibited by high salt and glycosaminoglycans (GAGs) heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate, but not by dextran or hyaluronan, indicating that the chemokines bind to highly sulfated GAGs. Chemokine binding to synovial structures correlated strongly with avidity of chemokine binding to heparin (SLC > TARC > RANTES > MIP1 beta > MCP1 > MIP1 alpha > FKN). A RANTES mutant with decreased avidity for heparin was not able to bind to ECM or mast cells. Thus, these data indicate that chemokines can bind to ECM and mast cell granule constituents in situ via interactions with GAGs. Further, only a subset of chemokines were able to bind efficiently to structures in the extravascular space, indicating that chemokines may form different types of gradients based on their GAG binding ability and that chemotactic gradients in tissues may be quite complex.
Asunto(s)
Artritis Reumatoide/inmunología , Quimiocinas/metabolismo , Fosfatasa Alcalina/metabolismo , Matriz Extracelular/metabolismo , Heparina/metabolismo , Humanos , Mastocitos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Membrana Sinovial/metabolismoRESUMEN
The pathogenesis of rheumatoid arthritis (RA) may be mediated by Th1-type T cells. Since chemokine receptors CXCR3 and CCR5 are preferentially expressed on Th1 cells, we tested the expression and regulation of several chemokines, including those that signal through CXCR3 (interferon-gamma-inducible protein of 10 kDa, IP-10, CXCL10; and monokine induced by interferon-gamma, Mig, CXCL9) and CCR5 (macrophage inflammatory protein (Mip)-1 alpha, CCL3; and Mip-1 beta, CCL4) in RA synovial fluids, synovial tissues, and blood. Synovial fluid (SF) protein levels of IP-10 (32.1 +/- 10.5 ng/ml), Mig (15.0 +/- 6.4 ng/ml), Mip-1 beta (0.7 +/- 0.3 ng/ml), and Mip-1 alpha (0.8 +/- 0.1 ng/ml) were 100-, 50-, 25-, and 2-fold elevated in RASF compared to control SF (P < 0.001, P < 0.001, P < 0. 001, and P < 0.02, respectively). Tissue levels of IP-10, Mig, and Mip-1 beta were significantly higher in RA than in OA (P < 0.01). Serum levels of IP-10 (3.1 +/- 1.2 ng/ml) were higher in patients with seropositive RA compared to controls (1.2 +/- 0.2 ng/ml) (P < 0.02). There was a gradient of IP-10, Mig, Mip-1 alpha, and Mip-1 beta from the blood into the synovial fluid in RA. Infiltrating T cells around high endothelial venules in RA synovium and 90 +/- 3% of SF CD3(+)CD4(+) T cells expressed CXCR3, and 85 +/- 2% of SF CD3(+)CD4(+) T cells expressed CCR5. Chemokines, including IP-10, Mig, Mip-1 alpha, and Mip-1 beta, may participate in the selective recruitment of CCR5(+)CXCR3(+) T cells to the inflamed synovium.
Asunto(s)
Artritis Reumatoide/metabolismo , Receptores CCR5/análisis , Receptores de Quimiocina/análisis , Membrana Sinovial/química , Quimiocinas/sangre , Humanos , Receptores CXCR3 , Linfocitos T/metabolismoRESUMEN
OBJECTIVE: To determine whether monocyte/macrophage expression of the CD6 ligand, activated leukocyte cell adhesion molecule (ALCAM) (CD166), is regulated by cytokines during inflammation in rheumatoid arthritis (RA). METHODS: We used flow cytometry to test whether cytokines present in rheumatoid synovium could regulate ALCAM cell surface expression on peripheral blood (PB) monocytes and RA synovial fluid (SF) macrophages, and we examined ALCAM expression in situ in RA synovium by immunofluorescence. RESULTS: The monocyte differentiation factors interleukin-3, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor augmented ALCAM expression on PB monocytes. ALCAM was expressed on monocyte-lineage cells in situ in inflamed synovium from patients with RA (9 of 9), but not in uninflamed synovium from patients with joint trauma (0 of 3). Furthermore, in vitro culture-induced ALCAM expression on PB monocytes and CD14+ RA SF cells was inhibited by an M-CSF neutralizing antibody. CONCLUSION: ALCAM expression on PB and SF monocytes/macrophages is enhanced by M-CSF.
Asunto(s)
Artritis Reumatoide/patología , Citocinas/farmacología , Monocitos/citología , Monocitos/inmunología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Linaje de la Célula/inmunología , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-2/farmacología , Interleucina-3/farmacología , Receptores de Lipopolisacáridos/biosíntesis , Factor Estimulante de Colonias de Macrófagos/farmacología , Persona de Mediana Edad , Pruebas de Neutralización , Líquido Sinovial/citologíaAsunto(s)
Células Epiteliales/fisiología , Nicotina/farmacología , Receptores Nicotínicos/biosíntesis , Linfocitos T/fisiología , Timo/fisiología , Transcripción Genética , Antígenos CD4/análisis , Antígenos CD8/análisis , Células Cultivadas , Humanos , Sustancias Macromoleculares , Neuronas/fisiología , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Subgrupos de Linfocitos T/fisiología , Linfocitos T/efectos de los fármacos , Timo/citologíaRESUMEN
Thymic dendritic cells (DCs) appear to have distinct biologic and functional properties compared with DCs in other tissues. Currently, little is known about human thymic DCs because they have been difficult to isolate and culture in vitro. Here, we report that human thymic stroma can support the development of primitive human hemopoietic stem cells into mature DCs without cytokine or serum supplementation. Coculture of CD34+CD38-lineage (lin)- and CD34+CD38+lin- umbilical cord blood cells with thymic stromal monolayers induced 43 +/- 17-fold and 32 +/- 16-fold expansions, respectively, of umbilical cord blood progenitors and also generated large numbers of cells with the morphologic, phenotypic, and functional characteristics of mature DCs. These cells expressed class I and class II MHC, CD1a, CD2, CD4, CD11c, CD40, CD45, CD80, CD83, and CD86 and were potent stimulators of allogeneic T cell activation. Primitive hemopoietic progenitors also developed into mature DCs in a novel tissue culture system of thymic nodules wherein thymic epithelial cells and fibroblasts were grown in nodular aggregates in vitro. These results demonstrate that human thymic stroma efficiently supports the development of CD34+CD38-lin- cord blood cells into mature DCs. In addition, the culture conditions described in this report are useful systems for studying the ontogeny of human DCs in thymic microenvironments.
Asunto(s)
Antígenos CD34 , Antígenos CD , Antígenos de Diferenciación , Células Dendríticas/citología , Sangre Fetal/inmunología , NAD+ Nucleosidasa , Timo/citología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Antígenos CD1/biosíntesis , Antígenos CD34/biosíntesis , Antígenos de Diferenciación/biosíntesis , Diferenciación Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Epiteliales/citología , Células Epiteliales/inmunología , Sangre Fetal/citología , Fibroblastos/citología , Fibroblastos/inmunología , Humanos , Inmunofenotipificación , Receptores de Lipopolisacáridos/biosíntesis , Activación de Linfocitos , Glicoproteínas de Membrana , NAD+ Nucleosidasa/biosíntesis , Células del Estroma/citología , Células del Estroma/inmunología , Timo/inmunología , Timo/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Thymic tissues express transcripts encoding the alpha-3, alpha-5 and beta-4 subunits of nicotinic neuronal acetylcholine receptors (AcChRs) suggesting that neuronal AcChRs similar to those expressed in ganglia are expressed in the thymus. Transcription occurs in both isolated thymocytes and thymic epithelial cells. RT-PCR analyses of thymocyte subsets indicate that immature CD4 + 8 + thymocytes express higher levels of the alpha-3 and beta-4 transcripts than more mature thymocytes. Compared to freshly isolated thymocytes, peripheral blood lymphocytes do not express alpha-3 and beta-4 AcChR subunit transcripts. Cultured thymocytes rapidly down-regulate transcription of the alpha-3 and beta-4 AcChR subunit genes by a process that is not reversed by stimulation with phytohemagglutinin and IL-2. Thus our results indicate that there is transcriptional regulation of neuronal AcChR subunit genes during the process of thymocyte maturation and that factors within the thymic microenvironment influence expression of the alpha-3 and beta-4 AcChR subunit genes by developing T cells.
Asunto(s)
Neuronas/fisiología , Receptores Nicotínicos/genética , Timo/fisiología , Precursor de Proteína beta-Amiloide/genética , Antígenos CD4/análisis , Antígenos CD8/análisis , Células Cultivadas , Senescencia Celular , Células Epiteliales/fisiología , Humanos , Linfocitos/fisiología , Timo/citología , Timo/inmunología , Transcripción GenéticaRESUMEN
CD6 is a 130-kD glycoprotein expressed on the surface of thymocytes and peripheral blood T cells that is involved in TCR-mediated T cell activation. In thymus, CD6 mediates interactions between thymocytes and thymic epithelial (TE) cells. In indirect immunofluorescence assays, a recombinant CD6-immunoglobulin fusion protein (CD6-Rg) bound to cultured human TE cells and to thymic fibroblasts. CD6-Rg binding to TF and TE cells was trypsin sensitive, and 54 +/- 4% of binding was divalent cation dependent. By screening the blind panel of 479 monoclonal antibodies (mAbs) from the 5th International Workshop on Human Leukocyte Differentiation Antigens for expression on human TE cells and for the ability to block CD6-Rg binding to TE cells, we found one mAb (J4-81) that significantly inhibited the binding of CD6-Rg to TE cells (60 +/- 7% inhibition). A second mAb to the surface antigen identified by mAb J4-81, J3-119, enhanced the binding of CD6-Rg to TE cells by 48 +/- 5%. Using covalent cross-linking and trypsin digestion, we found that mAb J4-81 and CD6-Rg both bound to the same 100-kD glycoprotein (CD6L-100) on the surface of TE cells. These data demonstrate that a 100-kD glycoprotein on TE cells detected by mAb J4-81 is a ligand for CD6.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Glicoproteínas/aislamiento & purificación , Timo/química , Molécula de Adhesión Celular del Leucocito Activado , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Niño , Epitelio/química , Epítopos/inmunología , Fibroblastos/química , Glicoproteínas/química , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Humanos , Peso Molecular , Especificidad de Órganos , Proteínas Recombinantes de Fusión/metabolismo , Timo/citologíaRESUMEN
Cellular interactions between developing thymocytes and cells of the thymic microenvironment are necessary for maturation of thymocytes into mature T cells. While much is known about the molecules on developing T cells that mediate these interactions, little is known about the surface molecules of human thymic epithelial (TE) cells. In this study, using a panel of 276 MAb including 255 MAb from the 5th International Workshop on Human Leukocyte Differentiation Antigens (HLDA-V), we have determined the expression of CD1 through CDw130 and other surface molecules on resting and IFN-gamma-activated cultured human TE cells and on resting epidermal keratinocytes (EK). We demonstrate the surface expression of 50 of the 161 molecules assayed for on TE cells, including a number of adhesion molecules, cytokine receptors, Apo-1, and MHC-encoded molecules. While activation of TE cells with IFN-gamma for 48 hr induced a greater than fivefold increase in the expression of four surface molecules (CD38, CD54, MHC class I, and MHC class II), it also induced a greater than 50% increase in the expression of 14 other surface molecules (CD12, CD29, CD40, CD44, CD47, CD49b, CD49c, CD49e, CD55, CD66, CD87, CD104, TE4, and STE3) and a decrease in the expression of three molecules (CDw65, CDw109, and STE2). In comparing the phenotype of TE cells to 83 other cell lines studied in HLDA-V, we found that TE cells were strikingly more similar to EK than to any of the other cell types tested.
Asunto(s)
Antígenos de Diferenciación/inmunología , Epidermis/inmunología , Queratinocitos/inmunología , Timo/inmunología , Antígenos CD/inmunología , Células Epidérmicas , Humanos , Interferón gamma/farmacología , Fenotipo , Timo/citología , Timo/efectos de los fármacosRESUMEN
Interactions between T cells and epithelial cells of the thymus are essential for normal T cell development, and interactions between T cells and skin epidermal keratinocytes occur in the context of inflammatory skin diseases and cutaneous T cell malignancies. On the basis of observations that the T cell ALL cell line, HSB, bound to IFN-gamma activated epidermal keratinocytes (41 +/- 5% of EK with three or more HSB cells bound), whereas the CTCL cell line H9 bound poorly (8 +/- 3%), we have raised mAb 13H12 that identified a 36 kDa molecule, termed AD2, that was highly expressed on HSB but not on H9 T cells. mAb 13H12 inhibited the binding of HSB T cells to IFN-gamma-treated epidermal keratinocytes (43 +/- 5% inhibition, p < 0.01), inhibited the binding of peripheral blood T cells to IFN-gamma-treated EK (62 +/- 3% inhibition, p < 0.001), and inhibited the binding of IFN-gamma-treated human thymic epithelial cells to thymocytes (39 +/- 3% inhibition, p < 0.01). Although AD2 was expressed at a low level on all T cells, AD2 was highly expressed on the CD3-CD4-CD8-, CD3-CD4low+ CD8-, and CD3-CD4+ CD8+ subsets of immature thymocytes in the thymic subcapsular and inner cortex. Taken together, these data suggest a role for the AD2 molecule in interactions of T cells with epithelial cells of skin and thymus.
Asunto(s)
Queratinocitos/fisiología , Linfocitos T/fisiología , Timo/citología , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Antígenos de Diferenciación de Linfocitos T/metabolismo , Adhesión Celular/inmunología , Adhesión Celular/fisiología , Línea Celular , Células Epiteliales , Humanos , Interferón gamma/metabolismo , Queratinocitos/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/fisiología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/fisiología , Linfocitos T/inmunología , Timo/inmunología , Timo/fisiologíaRESUMEN
It has recently been recognized that CD44 comprises a large family of alternatively spliced forms. In the thymus, CD44 has been postulated to play an important role in immature T cell migration and maturation. In this paper, we have studied the expression of CD44 molecules and two CD44 ligands, hyaluronan (HA) and fibronectin (FN), during human thymic fetal development. We found that mAbs against all CD44 isoforms (A3D8 or A1G3) reacted with both thymic epithelial (TE) cells and thymocytes beginning at the time of initial colonization of the human thymus by hematopoietic stem cells at 8.2 weeks of fetal gestation. However, mAbs specific for splice variants of CD44 containing membrane-proximal inserts (11.24, 11.10 and 11.9) reacted only with terminally differentiated TE cells in and around Hassall's bodies beginning at 16-19 weeks of fetal gestation. Studies of differentiated versus undifferentiated TE cells in vitro confirmed the selective expression of CD44 variant isoforms on terminally differentiated TE cells. Expression of HA and FN was determined by fluorescence microscopy using either biotinylated-HA binding protein or an anti-FN mAb. We found that whereas FN was present throughout the human fetal thymus beginning at 8.2 weeks, HA was not present until 16 weeks of gestational age. These data demonstrate the differential expression of standard versus variant CD44 isoforms during thymic ontogeny and implicate CD44 interactions with ligands other than HA as important in the earlier stages of human thymus development.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/inmunología , Fibronectinas/biosíntesis , Ácido Hialurónico/biosíntesis , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/inmunología , Receptores Mensajeros de Linfocitos/biosíntesis , Receptores Mensajeros de Linfocitos/inmunología , Timo/embriología , Anticuerpos Monoclonales/inmunología , Diferenciación Celular , Células Cultivadas , Desarrollo Embrionario y Fetal/inmunología , Fibronectinas/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Humanos , Receptores de Hialuranos , Ácido Hialurónico/metabolismo , Recién Nacido , Timo/citologíaRESUMEN
Human thymic epithelial (TE) cells produce interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6, cytokines that are important for thymocyte proliferation. The mRNAs for these cytokines are short-lived and are inducible by multiple stimuli. Thus, the steady-state levels for IL-1 and IL-6 mRNAs are critical in establishing the final cytokine protein levels. In this study we have evaluated the effect of epidermal growth factor (EGF), a growth factor for TE cells, and its homologue transforming growth factor alpha (TGF-alpha), on primary cultures of normal human TE cells for the levels of IL-1 alpha, IL-1 beta, IL-6, and TGF-alpha mRNA. We showed that TE cells expressed EGF receptors (EGF-R) in vitro and in vivo, and that treatment of TE cells with EGF or TGF-alpha increased IL-1 and IL-6 biological activity and mRNA levels for IL-1 alpha, IL-1 beta, and IL-6. Neither EGF nor TGF-alpha increased transcription rates of IL-1 alpha, IL-1 beta, and IL-6 genes, but rather both EGF and TGF-alpha increased cytokine mRNA stability. By indirect immunofluorescence assay, TGF-alpha was localized in medullary TE cells and thymic Hassall's bodies while EGF-R was localized to TE cells throughout the thymus. Thus, TGF-alpha and EGF are critical regulatory molecules for production of TE cell-derived cytokines within the thymus and may function as key modulators of human T cell development in vivo.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Interleucina-1/genética , Interleucina-6/genética , ARN Mensajero/análisis , Timo/metabolismo , Factor de Crecimiento Transformador alfa/farmacología , Células Cultivadas , Cicloheximida/farmacología , Dactinomicina/farmacología , Epitelio/metabolismo , Receptores ErbB/análisis , Humanos , Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Transcripción GenéticaRESUMEN
The hallmark of infection with HIV-1 is progressive depletion and qualitative dysfunction of the CD4+ Th cell population in infected individuals. Clinical trials of antiretroviral agents have shown that, despite suppression of virus replication, regeneration of the T cell pool does not occur. One proposed explanation for the defective regenerative capacity of the CD4+ T cell pool is infection of early T lymphocyte progenitors or stem cells. An additional explanation could be failure of cells of the intrathymic microenvironment (thymic epithelial (TE) cells) to carry out critical nurturing functions for developing thymocytes, i.e., secretion of thymocyte-trophic cytokines and expression of adhesion molecules. This study examines the effect of HIV on cultured TE cells and determines the role of TE cells in the regulation of viral expression in chronically HIV-infected cells. We found no evidence of infection of TE cells after exposure to HIV-1. However, normal human serum induced secretion of IL-6 by TE cells; induction of TE IL-6 was partially blocked by anti-IFN-gamma antibodies. Moreover, supernatants from TE cells maintained in normal human serum up-regulated HIV replication in chronically HIV-1-infected cells. Because intrathymic T cell precursors can be infected with HIV and T cell precursors come into close contact with TE cells in the thymus, IL-6 secreted by TE cells during normal intrathymic development may induce HIV expression in infected thymocytes in vivo and promote the intrathymic spread of HIV.
Asunto(s)
VIH/fisiología , Interleucina-6/metabolismo , Timo/microbiología , Comunicación Celular , Células Cultivadas , Niño , Epitelio/metabolismo , Epitelio/microbiología , Humanos , Interferón gamma/fisiología , Timo/metabolismo , Regulación hacia Arriba , Replicación ViralRESUMEN
The development of conditions for culturing normal human thymic epithelial (TE) cells free from contaminating stromal cells has allowed us to characterize a number of cytokines produced by TE cells. Using cDNA probes for human IL-6, granulocyte-monocyte-CSF, and leukemia inhibitory factor (LIF), we identified mRNA for these cytokines by RNA blot analysis of total RNA preparations derived from TE cells. We demonstrated that TE cells produced IL-6 transcripts and that TE cell culture supernatants contained IL-6 biologic activity, as determined by the ability to support proliferation of the T1165 plasmacytoma line. The 1.0-kilobase (kb) transcript of granulocyte-monocyte-CSF was also detected in TE cell-derived total RNA. TE cell culture supernatants contained LIF activity, as determined by proliferation of the murine cell line DA-1a, and a 4.0-kb LIF transcript was detected in TE cell-derived total RNA preparations. The 4.0-kb LIF transcript from TE cell-derived total RNA corresponded in size to the LIF transcripts in PMA-activated T lymphocytes. Thus, using biologic assays and RNA blot analysis, we demonstrated that cultured normal human TE cells produced both immunoregulatory cytokines and cytokines that drive various differentiation stages of human hematopoiesis. Our findings support the hypothesis that TE cells may play a role in providing cytokines that are important for the proliferation and differentiation of hematopoietic precursor cells that migrate to the thymus during fetal and postnatal human thymic development.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Inhibidores de Crecimiento/biosíntesis , Interleucina-6/biosíntesis , Linfocinas/biosíntesis , Timo/metabolismo , Antígenos CD7 , Antígenos de Diferenciación de Linfocitos T/análisis , Células Cultivadas , Epitelio/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Interleucina-6/genética , Factor Inhibidor de Leucemia , Linfocinas/genética , ARN/análisis , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/citologíaRESUMEN
Interaction of T lymphocytes with other cell types is important for normal T-cell development and function. Recently, a number of adhesion molecules important in T-cell interactions with other cell types have been defined. In this paper we review the role of two adhesion pathways, CD2/LFA-3 and LFA-1/ICAM-1, in T-cell interactions with epithelial cells of the thymus and skin. While thymic epithelium-T-cell interactions were mediated by both the LFA-1/ICAM-1 pathway and the CD2/LFA-3 pathway, epidermal-T-cell interactions were mediated primarily by the LFA-1/ICAM-1 pathway. Although ICAM-1 was not expressed in vivo on epidermal keratinocytes in normal skin, ICAM-1 was expressed by epidermal keratinocytes at the site of T-cell infiltration in inflammatory dermatitis. ICAM-1 was expressed in vivo on thymic epithelium. Both LFA-3 and ICAM-1 were expressed on epithelial cells of thymus and skin early on in fetal ontogeny. These antigen-independent adhesion molecules play an important role in the cell-cell interactions associated with T-cell differentiation and function.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Comunicación Celular , Piel/citología , Linfocitos T/citología , Timo/citología , Animales , Antígenos de Diferenciación/fisiología , Antígenos de Superficie/fisiología , Antígenos CD58 , Epidermis/metabolismo , Células Epiteliales , Humanos , Antígeno-1 Asociado a Función de Linfocito , Complejo Mayor de Histocompatibilidad/fisiología , Glicoproteínas de Membrana/fisiología , Receptores de Adhesión de Leucocito/fisiología , Timo/metabolismoRESUMEN
We have investigated the binding in vitro of activated thymocytes to thymic epithelial (TE) cells, and studied the effect of up-regulation of TE cell surface intracellular adhesion molecule 1 (ICAM-1) and HLA-DR by IFN-gamma on the ability of TE cells to bind to both resting and activated human thymocytes. TE cell binding to activated and resting thymocytes was studied by using our previously described suspension assay of TE-thymocyte conjugate formation. We found that activated mature and immature thymocytes bound maximally at 37 degrees C to IFN-gamma-treated ICAM-1+ and HLA-DR+ TE cells and this TE-activated thymocyte binding was inhibited by antibodies to LFA-1 alpha-chain (CD11a) (68.1 +/- 5.6% inhibition, p less than 0.01) and ICAM-1 (73.9 +/- 7.7% inhibition, p less than 0.05). Neither anti-HLA-DR antibody L243 nor anti-MHC class I antibody 3F10 inhibited IFN-gamma-treated TE binding to activated thymocytes. As with antibodies to LFA-3 and CD2, antibodies to LFA-1 and ICAM-1 also inhibited PHA-induced mature thymocyte activation when accessory signals were provided by TE cells in vitro. Finally, LFA-1 and ICAM-1 were expressed early on in human thymic fetal ontogeny in patterns similar to those seen in postnatal thymus. Taken together, these data suggest that resting mature and immature thymocytes bind to TE cells via the CD2/LFA-3 ligand pair, whereas activated thymocytes bind via both CD2/LFA-3 and LFA-1/ICAM-1 ligand systems. We postulate that IFN-gamma produced intrathymically may regulate TE expression of ICAM-1 and therefore potentially may regulate TE cell binding to activated thymocytes beginning in the earliest stages of human thymic development.
Asunto(s)
Antígenos de Diferenciación/fisiología , Moléculas de Adhesión Celular/fisiología , Receptores de Adhesión de Leucocito/fisiología , Linfocitos T/citología , Timo/citología , Anticuerpos Monoclonales , Antígenos de Superficie/análisis , Antígenos CD58 , Adhesión Celular , Células Epiteliales , Técnica del Anticuerpo Fluorescente , Antígenos HLA-DR/inmunología , Humanos , Técnicas Inmunológicas , Técnicas In Vitro , Molécula 1 de Adhesión Intercelular , Interferón gamma/farmacología , Activación de Linfocitos , Antígeno-1 Asociado a Función de Linfocito , Glicoproteínas de Membrana/análisis , Timo/crecimiento & desarrolloRESUMEN
A complement fixing IgM monoclonal antibody (1B10) that reacts with surface membrane molecules of human fibroblasts, tissue macrophages, and peripheral monocytes was produced. In Western blot analysis of detergent extracts of cultured human foreskin fibroblasts, antibody 1B10 detected protein bands of Mr 43,000 and 72-80,000. We used the 1B10 antibody with complement to eliminate most 1B10 positive nonepithelial cells from thymic epithelial (TE) cell cultures, thereby allowing us to grow highly enriched populations of human TE cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Separación Celular/métodos , Proteínas del Sistema Complemento/inmunología , Fibroblastos/citología , Antígenos/análisis , Western Blotting , Células Cultivadas , Citotoxicidad Inmunológica , Células Epiteliales , Epitelio/inmunología , Fibroblastos/inmunología , Humanos , Macrófagos/inmunología , Monocitos/inmunología , Piel/citología , Timo/citologíaRESUMEN
Synovitis in rheumatoid arthritis is characterized by infiltration of the synovium by T and B lymphocytes and monocytes, as well as by the proliferation of synovial lining cells, fibroblasts, and endothelial cells. To study synovial cell-T cell interactions in vitro, we established cultures of fibroblast-like synovial cells, and used these cells in a synovial cell-T cell binding assay. Using T cells at various stages of differentiation and activation, we found that human thymocytes and mitogen-activated peripheral blood T cells bound to fibroblast-like synovial cells, whereas fresh peripheral blood T cells did not. Moreover, activated T cells from inflammatory synovial tissue or from synovial fluid also bound to fibroblast-like synovial cells cultured in vivo. Antibodies against certain epitopes of the T cell CD2 (35.1) and synovial cell lymphocyte function-associated antigen-3 (LFA-3) (TS2/9) molecules inhibited synovial cell-thymocyte binding. However, these same anti-CD2 and anti-LFA-3 antibodies only partially inhibited synovial cell binding to activated normal peripheral blood T cells. Moreover, T cells from inflammatory synovium from rheumatoid arthritis and psoriatic arthritis patients also bound to synovial cells in vitro. These findings demonstrate that fibroblast-like synovial cells are capable of binding to human T cells in vitro, and suggest that during the course of inflammatory synovitis, synovial fibroblast-T cell interactions may occur in vivo.