RESUMEN
Increasing evidence suggests a role for the immune system in amyotrophic lateral sclerosis (ALS). To determine the extent of the immune activation in ALS we analyzed the expression and cellular distribution of components of innate and adaptive immunity in spinal cord (SC) and motor cortex (MCx) from patients with rapid and slow sporadic ALS and controls. High levels of mRNA and protein of classical complement pathway, C1q and C4, as well as the downstream complement components C3 and C5b-9 were found in all ALS samples. Furthermore, we found higher numbers of activated microglia, reactive astrocytes, dendritic cells (DCs) and CD8(+) T-cells in ALS than in control tissue. Rapid ALS cases had more dendritic cells than slow ALS cases, whereas slow ALS cases had more activated microglia than rapid cases. Our findings demonstrate a persistent and prominent activation of both innate and adaptive immunity in ALS. We propose a complement-driven immune response which may contribute to the progression of the inflammation and ultimately lead to even more motor neuron injury.
Asunto(s)
Inmunidad Adaptativa/inmunología , Esclerosis Amiotrófica Lateral/inmunología , Activación de Complemento/inmunología , Inmunidad Innata/inmunología , Adulto , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Análisis de Varianza , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Inflamación/inmunología , Inflamación/metabolismo , Masculino , Microglía/inmunología , Microglía/metabolismo , Persona de Mediana Edad , Neuronas Motoras/inmunología , Neuronas Motoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Médula Espinal/inmunología , Médula Espinal/metabolismoRESUMEN
Type-1 cytokine-producing T cells are important in the pathogenesis of psoriasis vulgaris, for which efficient therapy is provided by means of narrow-band ultraviolet-B (NB-UVB). The expression of the type-1 cytokine interferon-gamma (IFN-gamma) is regulated by interleukin-12 (IL-12), IL-15, IL-18 and IL-23; however, not much is known about the effect of this therapy on the levels of these cytokines in lesional psoriatic skin in situ. In this study, we investigated the effects of NB-UVB therapy on the expression of IFN-gamma-inducing cytokines. Ten patients with chronic plaque-type psoriasis selected to be treated with NB-UVB therapy were recruited for these experiments and the expression of cytokines IL-12, IL-15, IL-18, IL-23 and IFN-gamma in lesional psoriatic skin before, during and after therapy was determined with the help of immunohistochemistry. Double staining was performed in order to determine the cell types expressing these cytokines. The decrease in the psoriasis area and severity index was accompanied by a significant decrease in the expression of IFN-gamma, and concomitantly, significant reduction of IFN-gamma inducers -- IL-12, IL-18 and IL-23. Thus, we concluded that the decrease of IFN-gamma expression in psoriasis lesions after NB-UVB therapy could be a result of diminished expression of IL-12, IL-18 and IL-23 in lesional skin. Therapies targeting these three cytokines should, therefore, be considered in the treatment of psoriasis.
Asunto(s)
Interferón gamma/metabolismo , Interleucina-12/biosíntesis , Interleucina-18/biosíntesis , Interleucinas/biosíntesis , Psoriasis/metabolismo , Psoriasis/radioterapia , Rayos Ultravioleta , Adulto , Femenino , Humanos , Inmunohistoquímica , Interleucina-23 , Subunidad p19 de la Interleucina-23 , Masculino , Persona de Mediana EdadRESUMEN
Normal human skin shows preferential (epi)dermal infiltration of CD4+ T cells upon acute UV exposure. To study the mechanism behind this feature we locally exposed healthy volunteers to doses of UV commonly encountered by the population. Expression of integrins on T cells and expression of adhesion molecules on dermal endothelial cells were quantitatively assessed by immunohistochemistry in situ. We also investigated the effects of ultraviolet-B (UVB) exposure on psoriasin and IL-16, two specific chemoattractant factors for CD4+ T cells, at messenger RNA (mRNA) level by semiquantitative reverse transcriptase-polymerase chain reaction and at protein level by immunohistochemistry. We found, at day 2 after exposure to four minimal erythema doses of UVB, predominant accumulation of LFA-1+/CLA-/VLA-4- T cells in the dermis. Concomitantly the expression of ICAM-1, but not that of E-selectin and VCAM-1, was upregulated on dermal endothelial cells. The increase in the number of dermal T cells was not due to proliferation because only 2% of the UVB-induced dermal T cells expressed the marker of proliferation Ki-67. Whereas exposure to 35 J/cm2 of ultraviolet-A (UVA), like UVB, induced a loss of intraepidermal T cells at day 2 after exposure, UVA induced neither any influx of T cells into the dermis nor any adhesion molecule upregulation on endothelial cells. In response to UVB exposure, the expression of psoriasin mRNA, but not of IL-16 mRNA, was upregulated; the expression of psoriasin protein was also found to be upregulated. These results suggest that LFA-1/ICAM-1 pathway and psoriasin are both involved in the accumulation of CD4+ T cells into UVB-irradiated skin, possibly via a recruitment mechanism.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Quimiocinas/metabolismo , Piel/efectos de la radiación , Linfocitos T/efectos de la radiación , Rayos Ultravioleta , Secuencia de Bases , Cartilla de ADN , Humanos , Inmunohistoquímica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína A7 de Unión a Calcio de la Familia S100 , Proteínas S100 , Piel/citología , Piel/metabolismo , Linfocitos T/metabolismoRESUMEN
Acute, low-doses of ultraviolet (UV)-B radiation affect the immune competent cells of the skin immune system. In this study, we examined the time-dependent changes of the cutaneous T cell population in normal human volunteers following a single local exposure to UV. Solar-simulated UV radiation caused an initial decrease in intraepidermal T cell numbers, even leading to T cell depletion at day 4, whereupon a considerable infiltration of T cells in the epidermis occurred that peaked at day 14. In the dermis the number of T cells was markedly increased at days 2 (peak) and 4 after irradiation, and subsequently declined to the nonirradiated control values at day 10. Double-staining with several T cell markers showed that the T cells, infiltrating the (epi)dermis upon UV exposure, were almost exclusively CD4+ CD45RO+ T cells, expressing an alpha/beta type T cell receptor, but lacking the activation markers HLA-DR, VLA-1, and IL-2R. Application of UVB radiation resulted in similar dynamics of T cells, indicating that the UVB wavelengths within the solar-simulated UV radiation were responsible for the selective influx of CD4+ T cells. In conjunction with UVB-induced alterations in the type and function of antigen-presenting cells (i.e., Langerhans cells and macrophages), the changes of the cutaneous T cell population may also contribute to UVB-induced immunosuppression at skin level in man.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/efectos de la radiación , Piel/inmunología , Piel/efectos de la radiación , Rayos Ultravioleta , Adolescente , Adulto , Complejo CD3/análisis , Antígenos CD4/biosíntesis , Antígenos CD4/inmunología , Antígenos CD4/efectos de la radiación , Linfocitos T CD4-Positivos/citología , Antígenos CD8/biosíntesis , Antígenos CD8/inmunología , Antígenos CD8/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Femenino , Humanos , Memoria Inmunológica/efectos de la radiación , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/efectos de la radiación , Piel/citología , Luz Solar/efectos adversos , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/efectos de la radiación , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Factores de TiempoRESUMEN
In this study, we investigated the effect of ultraviolet B radiation on human Langerhans cell function. Normal human skin was irradiated ex vivo with single doses of ultraviolet B. For assessment of T-cell stimulatory function, cells that spontaneously migrated from epidermal sheets were used, whereas full-thickness skin biopsies were used to investigate alterations in migratory properties. The cells migrating from ultraviolet B-exposed epidermal sheets demonstrated a decrease in the percentage of HLA-DR positive Langerhans cells, as well as a reduced capacity to induce proliferation of allogeneic T cells, when compared with cells migrating from nonexposed sheets. When a correction was made for the decreased number of HLA-DR positive Langerhans cells migrating from ultraviolet B-exposed epidermis, however, it appeared that the capacity to induce T-cell proliferation was identical for Langerhans cells migrating from ultraviolet B-exposed and nonexposed epidermis. The presence of ultraviolet B-induced DNA damage could be demonstrated in the Langerhans cells from ultraviolet B-treated skin, indicating that the cells had received significant doses of ultraviolet B. As regards the effect of ultraviolet B on migratory properties of Langerhans cells, we found not only that reduced numbers of CD1a-positive Langerhans cells migrated from the ultraviolet B-exposed full-thickness skin, but also that there was a reduction in CD1a-positive Langerhans cells in the epidermis. This implies that ultraviolet B induces death of Langerhans cells as well as loss of cell surface molecules rather than altering Langerhans cells migration, whereas the Langerhans cells that were still able to migrate fully retained the capacity to activate allogeneic T cells.
Asunto(s)
Daño del ADN/efectos de la radiación , Antígenos HLA-DR/análisis , Células de Langerhans/fisiología , Células de Langerhans/efectos de la radiación , Piel/inmunología , Piel/efectos de la radiación , Rayos Ultravioleta , Células Presentadoras de Antígenos/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Relación Dosis-Respuesta en la Radiación , Humanos , Células de Langerhans/citología , Prueba de Cultivo Mixto de Linfocitos , Piel/citologíaRESUMEN
The capacity of APC to stimulate the proliferation of human peripheral blood T cells decreases upon ultraviolet-B (UVB) irradiation. The aim of this study was to investigate whether all T cell subsets are equally sensitive to this reduced APC function. Established human Th1, Th2, and Th0 clones were stimulated with monocytes in a soluble CD3 mAb-mediated assay that is dependent on the presence of APC. Monocytes were exposed to low nonlethal doses of UVB radiation before coculture with T cells. UVB irradiation inhibited the capacity of monocytes to stimulate the proliferation and IFN-gamma production of Th1 cells in a dose-related fashion. In contrast, UVB-treated monocytes induced normal proliferation and IL-4 production in Th2 cells. Stimulation of Th0 cell proliferation by UVB-irradiated monocytes was normal, but a preferential suppression of IFN-gamma production was observed, thus leading to a more Th2-like cytokine response. The loss of Th1 proliferation upon stimulation with UVB-irradiated monocytes could be overcome by rIL-2; however, IFN-gamma production remained suppressed. IFN-gamma production could be completely restored by rIL-12, whereas the addition of IL-1 beta, TNF-alpha, or indomethacin had no such effect, nor did the addition of mAb to CD28, added to compensate for the reduced B7 expression of UVB-irradiated monocytes. Monocytes exposed to UVB radiation exhibited reduced expression of mRNA for the IL-1 2 subunits p35 and p40 and suppressed production of the IL-12 p70 protein. Our results thus indicate that UVB irradiation of APC selectively impairs Th1-like responses, a phenomenon caused by the UVB-induced suppression of monocyte IL-12 production.
Asunto(s)
Terapia de Inmunosupresión , Interleucina-12/biosíntesis , Interleucina-12/efectos de la radiación , Activación de Linfocitos/efectos de la radiación , Monocitos/metabolismo , Monocitos/efectos de la radiación , Células TH1/inmunología , Células TH1/efectos de la radiación , Rayos Ultravioleta , Citocinas/biosíntesis , Humanos , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interferón gamma/efectos de la radiación , Interleucina-2/farmacología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Células Th2/efectos de la radiaciónRESUMEN
Purified peripheral blood human T lymphocytes, derived from normal individuals, were assayed for their susceptibility to low doses of ultraviolet B (UVB) in vitro. Exposure of T cells to graded single doses (range 0-8 mJ/cm2) of UVB resulted in a dose-dependent reduction of viability. This phototoxic effect was not immediately apparent, however, but became manifest 48-72 h subsequent to irradiation. A dose as little as 0.5-1 mJ/cm2 was sufficient to cause 50% mortality. Irradiated T cells showed a reduced ability to proliferate, irrespective of the stimulus used, and a reduced ability to produce cytokines IL-2, IL-4, IL-5, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). This decreased ability was UVB-dose related and, remarkably, was exactly correlated to phototoxicity. UVB had no effect on CD4 and CD8 expression or their ratio, whereas the expression of IL-2R (CD25) was only slightly reduced. Our data suggest that UVB radiation neither selectively affects Th1 or Th2 nor CD4 or CD8 T cell subsets. The high susceptibility of T cells to UVB might explain, at least in part, the beneficial effect of phototherapy during treatment of certain immunodermatological diseases.
Asunto(s)
Linfocitos T/efectos de la radiación , Rayos Ultravioleta , Antígenos CD4/análisis , Antígenos CD8/análisis , Supervivencia Celular/efectos de los fármacos , Citocinas/biosíntesis , Humanos , Técnicas In Vitro , Activación de Linfocitos/efectos de la radiación , Receptores de Interleucina-2/análisis , Linfocitos T/inmunologíaRESUMEN
A method for the concentration of cells from cerebrospinal fluid is described. An adaptation of a commercial cytochamber, consisting of a holder that fixes a disposable chamber directly on a microscope slide, was used. The cells were spun down in a conventional swing-out centrifuge, which was provided with a bucket for the cytochamber system. After removing most of the supernatant with a pipette, the remaining fluid was absorbed by means of a suction device consisting of a disposable pipette tip covered with a piece of Leukopor and filled with Sephadex G10 beads. The method gives a high recovery of cells (90%), together with a good preservation of cell morphology, and leaves about 80% of the fluid available for analysis of the soluble components.
Asunto(s)
Líquido Cefalorraquídeo/citología , Centrifugación , Líquido Cefalorraquídeo/análisis , Humanos , Recuento de LeucocitosRESUMEN
The tissue factor activity of human brain thromboplastin and 6 commercial thromboplastins was determined by a spectrophotometric method and a two-stage coagulation assay. The thromboplastins were incubated with an excess of Factor VII and Factor X, and the activation of Factor X was estimated from the rate of hydrolysis of the chromogenic substrate S-2222 and from the coagulation time of plasma enriched in phospholipids. The results obtained by the two methods were related linearly and showed a correlation coefficient of 0.89. The coefficient of variation was 11% in the spectrophotometric method and 25% in the two-stage coagulation assay.