E. Garrido

Vasoactive intestinal peptide (VIP) mRNA expression in rat T and B lymphocytes.

Different evidence suggests that VIP has immunoregulatory functions and may be secreted by different cells involved in inflammatory and immune responses. In the present study, we demonstrate by reverse transcription (RT) and polymerase chain reaction (PCR) VIP gene expression in rat thymocytes and T and B cells derived from spleen and lymph nodes. We have obtained a specific VIP cDNA product of 458 bp identical in size to that obtained from cerebral cortex. These results have been confirmed by Southern blot analysis. VIP message has also been detected in the T-T hybridoma YH-1633 and in a non-immune cell line, the pheochromocytoma PC 12. VIP gene expression in central and peripheral lymphoid organs suggests that VIP may be a T and B cell-derived cytokine involved in T-cell differentiation and in cell immune responses.

Characterization of gene expression of VIP and VIP1-receptor in rat peritoneal lymphocytes and macrophages

In the present report we show the gene expression pattern of VIP and VIP1 receptor in two peritoneal cell populations, macrophages and lymphocytes by reverse transcription (RT) and polymerase chain reaction (PCR). Only in the lymphoid cells we have obtained a specific VIP cDNA product of 458 bp identical in size to the one obtained from cerebral cortex. On the other hand, we have obtained in both peritoneal populations lymphocytes and macrophages, a specific VIP1 receptor cDNA product of 311 bp identical in size to that obtained from lung. These results have been confirmed by Southern blot hybridization. Our findings suggest an autocrine/paracrine action of VIP in peritoneal microenvironment, supporting an immunoregulatory role for this neuropeptide.

Vasoactive intestinal peptide modulation of adherence and mobility in rat peritoneal lymphocytes and macrophages.

In this work, the effects of vasoactive intestinal peptide (VIP) in a concentration range from 10(-13) to 10(-7) M were studied in vitro on two common activities of peritoneal rat lymphocytes and macrophages: adherence and mobility (spontaneous and chemotaxis). The results show that VIP stimulated the adherence of the two cells studied, and increased the macrophage mobility but decreased this activity in lymphocytes. Moreover, a specific protein kinase C (PKC) activator such as phorbol myristate acetate (PMA, 50 ng/ml) also stimulated significantly the adherence and chemotaxis of both macrophages and lymphocytes. By contrast, a PKC inhibitor, retinal (2 x 10(-5) M), decreased significantly these capacities. Macrophages incubated with both VIP and PMA in relation to those incubated with VIP or PMA showed an increase in adherence and chemotaxis, whereas in lymphocytes adherence was also increased but chemotaxis decreased. The incubation with forskolin (10(-5) M), an enhancer of intracellular cAMP levels, produced an inhibitory effect of the chemotaxis activity in both types of cells. VIP prevented this inhibitory effect of forskolin in macrophages but not in lymphocytes. In addition, VIP was chemoattractant for macrophages but not for lymphocytes. The present study proves that VIP proves that VIP has a coronary effect on the two principal and representative types of immune cells in the rat peritoneum: lymphocytes and macrophages, stimulating macrophage chemotaxis through PKC activation and inhibiting lymphocyte chemotaxis through adenylate cyclase activation.

Differential VIP and VIP1 receptor gene expression in rat thymocyte subsets

Nervous, endocrine, and immune systems share a large number of regulatory molecules including hormones and neuropeptides. Vasoactive intestinal peptide (VIP) plays an important role in a variety of immunological functions. In the present report, we sorted purified thymocytes of the four major thymic subsets defined by CD4 and CD8 phenotypes. We demonstrate by reverse transcription (RT) and polymerase chain reaction (PCR) both VIP and VIP1 receptor gene expression in double positive (CD4+CD8+) and single positive (CD4+CD8-, CD4-CD8+) thymocyte subsets. Double negative thymocytes (CD4-CD8-) lack VIP and VIP1 receptor gene expression.

Pituitary adenylate cyclase-activating polypeptide (PACAP38) modulates lymphocyte and macrophage functions: stimulation of adherence and opposite effect on mobility

The effects of pituitary adenylate cyclase-activating polypeptide (PACAP38) in a concentration range from 10(-13) to 10(-6) M were studied, in vitro, on two functions of peritoneal rat lymphocytes and macrophages: adherence and mobility (spontaneous and chemotaxis). The results show that PACAP38 raised the adherence of the two cell types, increased the mobility of macrophages and decreased the mobility of lymphocytes. The maximal effects were observed at 10(-10) M in macrophages and at 10(-9) M in lymphocytes. Moreover, incubation with increasing concentrations of phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, resulted in a progressive enhancement of adherence and chemotaxis of both macrophages and lymphocytes. In contrast, retinal, a PKC inhibitor, significantly decreased these capacities. Incubation of macrophages with both PMA and PACAP38 did not have a synergistic effect on chemotaxis and adherence whereas, with lymphocytes, adherence was increased and chemotaxis was partially decreased. On the other hand, incubation with forskolin (an enhancer of intracellular cyclic AMP [cAMP] levels) caused inhibition and stimulation of chemotaxis and adherence, respectively, in both cell types. PACAP38 prevented the inhibitory effect of forskolin on chemotaxis of macrophages but not of lymphocytes, whereas the simultaneous presence of PACAP38 and forskolin was synergistic for adherence of both peritoneal cells. In addition, PACAP38 was chemoattractant for macrophages but not for lymphocytes. Furthermore, a VIP receptor antagonist was able to partially reverse the modulatory effects of PACAP38 on lymphocytes, but not on macrophages. These data suggest that PACAP38 exerts its action through the binding to type I PACAP receptors and PKC activation in macrophages and through the elevation of intracellular cAMP levels by binding to type II PACAP receptors in lymphocytes. The present work reveals an additional link between neuropeptides and the immune system and suggests that the peptide PACAP modulates the immunological function of macrophages and lymphocytes.

Lymphoid cell subpopulations containing vasoactive intestinal peptide in the rat

In the present study we describe the cell types containing immunoreactive vasoactive intestinal peptide (IR-VIP) in rat thymus, spleen, and lymph nodes. Indirect immunofluorescence staining and flow cytometry indicated that all lymphoid organs studied contained VIP-positive cells, with the spleen and lymph nodes having a higher proportion than the thymus. Vasoactive intestinal peptide was found in both lymphocytes and nonlymphoid cells, lymphocytes predominating among VIP-positive cells. Double immunofluorescent staining and flow cytometry showed that all lymphoid subpopulations identified contained variable proportions of VIP-positive lymphocytes. Immunocytochemical staining of cell suspensions for both light and electron microscopy showed the cytoplasmic localization of the IR-VIP. These findings, coupled to our previous results, are consistent with the idea that VIP may have a lymphoid origin and could be active in local immune responses.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP-38) Stimulates Rat Peritoneal Macrophage Functions

The present study shows that PACAP-38, in a dose-dependent manner, increased in vitro two steps of the phagocytic process in rat peritoneal macrophages: ingestion of inert particles (latex beads) and production of superoxide anion as measured by nitroblue tetrazolium reduction. The most effective concentration of PACAP-38 was 10(-10) M. Similarly, PMA, an activator of protein kinase C (PKC), increased the phagocytic activity in a dose-dependent manner, whereas retinal, a PKC inhibitor, decreased the activity. Macrophages incubated with forskolin, an enhancer of intracellular cAMP levels, produced an inhibitory effect on both phagocytic functions. The maximum stimulation of the phagocytic activity by PACAP-38 was not further enhanced by addition of PMA, suggesting that the enhancement of the phagocytic activity by PACAP-38 and PMA is mediated by a common signaling pathway. In addition, retinal as well as forskolin inhibited partially the stimulatory effect that PACAP-38 produced in the macrophage functions studied. Furthermore, this study showed that a VIP receptor antagonist was unable to suppress the stimulatory effect of PACAP-38. These results could prove that PACAP-38 stimulates the phagocytosis and production of superoxide anion in macrophages through PKC activation by binding to type I PACAP receptor.

EFFECTS OF DEXAMETHASONE ON THE LYMPHOID ORGANS OF RANA PEREZI

Owing to the possible importance of steroids in the mutual neuroendocrine-immune influences found in lower vertebrates, we study the structural and morphometrical changes induced by a single dose of dexamethasone, a synthetic corticosteroid, on the lymphoid organs of the frog Rana perezi. The dexamethasone treatment produces thymic involution with massive destruction of cortical lymphocytes, intense peripheral lymphopenia with lymphocyte redistribution to the bone marrow from peripheral blood and spleen. The effects on the spleen were less dramatic than in the thymus, but, the proportion of white pulp underwent a significant decrease and the size of splenic lymphoid follicles diminished. Our results demonstrate that Rana perezi is a corticosensitive species although the induced corticosteroid effects were less drastic than those described in other vertebrates, mainly mammals.

Erythropoiesis in the thymus of the spotless starling, Sturnus unicolor

SummaryAlthough previously described in other avian species, intrathymic erythropoiesis is a remarkable feature of the thymus of Sturnus unicolor. In discrete stages of the life cycle of this species, erythroblasts and mature erythrocytes occupy large areas of the thymic cortex and cortico-medullary border. Simultaneously, degenerated thymocytes and epithelial-reticular cells occur in the same areas. The relationship between intrathymic erythropoiesis, degeneration of cortical lymphocytes and epithelial-reticular cells, and macrophage activity is discussed and related to a possible functional role of sex hormones in this phenomenon.

Ultrastructural changes in the thymus of the turtle Mauremys caspica in relation to the seasonal cycle

SummaryChanges in the ultrastructure of the thymus of the turtle Mauremys caspica, with special reference to its non-lymphoid components, were studied in relation to the seasonal cycle. The thymic cortex contains framework-forming epithelial-reticular cells and free macrophages, while the medulla includes, in addition, mature and presumptive pro-interdigitating cells. The ultrastructural features of these cells are generally similar to those described for non-lymphoid components of the mammalian thymus. The turtle thymus undergoes cortical involution in spring, with recovery periods in May–June and during autumn. A moderate involution occurs in winter. At the beginning of spring, cortical (but not medullary) epithelial-reticular cells show degenerative changes, probably related to high levels of circulating testosterone. In spring and autumn, mature interdigitating cells are absent, but macrophages, monocytes, and pro-interdigitating cells are found. During May–June, the cortical epithelial-reticular population recovers and macrophages, monocytes, and interdigitating cells are actively phagocytic. In summer, the epithelial-reticular cells in both cortex and medulla display normal ultrastructural features; mature and immature interdigitating cells are absent and some macrophages are detected occasionally. The results suggest that non-lymphoid components of the reptilian thymus can play a role in governing T-lymphocyte differentiation, and that the thymic cortex and medulla exhibit different cycles of seasonal activity.