Alberto Durántez

Regulation of natural killer cytotoxicity by 1,25-dihydroxyvitamin D3.

The steroid hormone 1 alpha, 25-dihydroxyvitamin D3, calcitriol, is crucial in calcium homeostasis. Calcium plays a central role in T, B, and NK cell functions, and calcitriol is a known inhibitor of T cell proliferation and immunoglobulin production. We have analyzed here the immunoregulatory effects of calcitriol on NK cell function. We show that calcitriol specifically specifically inhibits, in a time- and dose-dependent fashion, the generation of cytotoxic activity from cultured CD16+ peripheral blood NK cells. It also suppresses, at similar molar concentrations (1-10 nM), interleukin 2 (IL-2) production by PHA-activated peripheral blood lymphocytes. Calcitriol does not interfere with the cytotoxic function of NK cells, whether fresh or generated in vitro, placing the inhibition at the level of NK cell activation. Interestingly enough, exogenous IL-2 can completely reverse the suppressive effect. These findings suggest that modulation of NK cell activation by control of the internal level of IL-2 may reflect an additional paracrine calcitriol-dependent circuit with immunoregulatory consequences.

Interleukin-2 induces cytotoxic activity in lymphocytes from regional axillary nodes of breast cancer patients.

Natural killer (NK) cells and lymphokine‐activated killer (LAK) cells have been involved in immuno‐surveillance against tumors. A normal NK activity was observed in peripheral blood (PB) mononuclear cells (MNC) from women with breast cancer, but a very low or absent NK cytotoxicity was found in the regional lymph node (RLN) MNC. However, strong cytotoxic activity against NK‐resistant and NK‐sensitive target cells can be induced in RLN MNC by long‐term (5‐day) incubation with recombinant interleukin‐2 (rIL‐2). This cytotoxic inducer effect of rIL‐2, not observed with recombinant interferon γ, was dose and time‐dependent and was not associated with modifications in the low number of Leu 11+ or Leu 7+ cells present in the population. Both the lack of NK activity and the generation of rIL‐2‐activated killer cells can be readily demonstrated in either histologically affected or unaffected RLN. These results stress the value of the immunomodulators inducing cytotoxic activity in RLN MNC of patients with tumors, and are discussed in association with their possible therapeutical role.

Functional impairment of natural killer cells in active ulcerative colitis: reversion of the defective natural killer activity by interleukin 2.

We have studied the functional characteristics and clinical importance of the natural killer (NK) cytotoxicity of peripheral blood mononuclear cells (PBMNC) from patients with ulcerative colitis. Normal NK activity was observed in PBMNC from patients with inactive disease, but a pronounced decrease was found in those with active disease. Clinical change from active to inactive disease was associated with enhancement of the depressed NK activity. The impairment of NK cytotoxicity found in patients with active disese could not be ascribed to a deficient number of NK cells as the amounts of HNK-1+, CD16+ (Leu 11), and CD11b (OKM1) cells in PBMNC were within normal ranges. This defective cytotoxic PBMNC activity was normalised by short term (18 hour) incubation with recombinant interleukin 2 (rIL-2). Moreover, long term (5 day) incubation of these effector cells with rIL-2 induced strong cytotoxic activity against NK resistant and NK sensitive target cells in patients with active and inactive disease. We also found that both precursors and effectors of cytotoxic activity promoted by short term and long term incubation with rIL-2 of PBMNC from the patients showed the phenotype of NK cells (CD16+, CD3-). Taken together, these results show that active ulcerative colitis is associated with a defective function of NK cells that is found to be normal in the inactive stage of the disease. The possible pathogenic and therapeutic implications of these findings are discussed.

Clinical signification of natural killer activity in B-cell chronic lymphocytic leukemia

The natural killer (NK) activity of peripheral blood mononuclear cells (PBMC) and lymphocytes with the capacity to form stable rosettes with neuraminidase‐treated sheep red blood cells (E+) was studied in 28 previously untreated patients (11 at stage 0, 10 at stage I and 7 at stages II and III, according to Rais classification) and 7 treated patients with B‐cell chronic lymphocytic leukemia (B‐CLL), all of them at stage 0 according to Rais classification after treatment, and in 15 healthy controls. The mean NK activities of PBMC and E+ lymphocytes from untreated patients were significantly decreased (p < 0.001) when compared with those of PBMC and E+ lymphocytes, respectively, from healthy controls. However, PBMC and E+ cells from treated patients demonstrated NK activity similar to that of the corresponding cellular populations of controls (p > 0.05). Furthermore, there were no significant differences among the NK activities of E+ lymphocytes from untreated B‐CLL patients in the different clinical stages 0, I, II and III, according to Rais classification (p > 0.05). These results demonstrate that the very low or undetectable levels of NK activity present in PBMC and E+ cell populations from previously untreated patients with B‐CLL, regardless of the clinical stage of the disease, can be modified by systemic therapy with alkylating agents. Moreover, the NK activity of PBMC and E+ lymphocytes from some treated patients that have achieved the stage 0 according to Rais classification after chemotherapy can be found within the range of the lytic activity shown by PBMC and E+ cells from normal donors.

Increased IgM B cell differentiation lymphokine production by T lymphocytes from patients with primary biliary cirrhosis

Hypergammaglobulinemia, mainly due to increased serum immunoglobulin M concentrations, is a common and distinctive feature of primary biliary cirrhosis. T-B cell cooperation plays a pivotal role in the regulation of immunoglobulin secretion. In this paper, the production of regulatory B lymphokines by T cells, as well as the functional response of B lymphocytes to these molecules, was investigated in patients with primary biliary cirrhosis. T cells from patients with primary biliary cirrhosis have an enhanced ability to produce lymphokines that regulate the proliferation of B cells and their differentiation to immunoglobulins G- and M-secreting cells. In contrast, the cellular production of lymphokines involved in the induction of immunoglobulin A-secreting cells was normal. Simultaneously, the proliferation and differentiation of purified B cells in response to stimulation with surface immunoglobulin ligands and lymphokines were normal. These results suggest that the elevated serum levels of immunoglobulins M and G found in patients with primary biliary cirrhosis could be ascribed to an enhanced lymphokine-mediated T-B cooperation.

Late relapse with nodular lymphoma after treatment for diffuse non-Hodgkin's lymphoma

Histologic conversion from a low‐grade non‐Hodgkins lymphoma (NHL) into a more aggressive histologic pattern is a common, well‐documented event in NHL. The converse phenomenon, appearance of a low‐grade, follicular NHL after treatment for diffuse, intermediate, or high‐grade NHL, has only recently been recognized. The clinical, morphologic, and immunologic features of a patient in whom relapse with an indolent nodular lymphoma was noticed after combination chemotherapy for diffuse lymphoma are presented. Immunologic markers at presentation and relapse were similar. Other previously reported cases are reviewed. Implications for diagnosis and therapy as well as the pathogenesis of this unique form of conversion are discussed.

Proliferation of B Cells from Chronic Lymphocytic Leukemia Is Selectively Promoted by B Cell Growth Factor

B cell activation was studied in B cells from B cell chronic lymphocytic leukemia (B-CLL) patients. After in vitro stimulation, these B cells showed extensive proliferation in the presence of high-molecular-weight B cell growth factor (BCGF). In contrast, this effect was not observed upon addition of recombinant interleukin-2 (IL-2). In agreement, upon stimulation, B cells expressed Bac-1 antigen but failed to acquire the IL-2 receptor. These results demonstrate that the utilization of the BCGF pathway can be segregated from that of IL-2 in B cells from B-CLL patients.

Natural Killer Cell Proliferation and Renal Disease: A Functional and Phenotypic Study

We describe a 57-year-old woman who presented with a constitutional syndrome, glomerulonephritis, and lymphocytosis. The phenotypic study, using flow cytometry, showed an expansion of natural killer (NK) cells (CD2+, CD3-, CD16+, CD56+, and CD7+). We performed a functional study of peripheral blood mononuclear cells (PBMCs) and of purified CD16+ cells (NK cells) and CD3+ cells (normal T cells). The expanded NK cell population, CD16+, did not proliferate with phytohemagglutinin (PHA) or anti-CD3 but showed a dose-dependent proliferation with recombinant interleukin-2 (rIL-2) and also proliferated with phorbol dibutyrate. This population showed very strong NK and lymphokine-activated killer cell (LAK) activities. The patients symptoms resolved spontaneously without treatment. Three years later, however, there is still abnormal renal function, and the expansion of NK cells persists, although with no indication of malignancy. We review the features of the different large granular lymphocyte proliferations and their seldom described relationship with renal disease.

Functional and Phenotypic Analysis of T-lymphocytes in Laryngeal Carcinoma

We studied the functional response and phenotypic characterization of peripheral blood T cells and their correlation with the clinical stage of disease in 29 males with previously untreated carcinoma of the larynx and 24 healthy male controls. Peripheral blood T cells, phenotypically CD2+ CD3+, were significantly decreased in the patients relative to the controls. Patients with advanced locoregional extension (T4 and N1, 2, 3) also showed a diminution of the CD4+ subpopulation of T cells. DNA synthesis by purified T cells showed similar blastogenic responses in patients and controls; the interleukin-2 production of phytohemagglutinin stimulated lymphocytes was also normal. We conclude that in patients with laryngeal carcinoma there is a phenotypic alteration of the T cells that is variable according to tumor stage, without functional alterations in blastogenic capacity or IL-2 production.

Characterization of adrenal medullary chromaffin cells by flow cytometry

BACKGROUND Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.