Induction of interleukin-15 production by HIV-1 nef protein: a role in the proliferation of uninfected cells.

Several recent reports have provided evidence that Nef enhances human immunodeficiency virus HIV infectivity, and in vitro experiments with the nef gene have demonstrated the possible role of Nef in modulating immune responses. Exogenous Nef has been demonstrated to induce proliferation of normal human peripheral blood mononuclear cells (PBMC) and to enhance HIV-1 replication. The aim of this study was to evaluate the biological mechanisms by which Nef, used as exogenous protein, modulates cellular activation. We showed that exogenous Nef protein induces the proliferation of unstimulated and suboptimally stimulated normal human PBMC, while it has no effect on the proliferation of optimally stimulated PBMC. Moreover, the activating effect of exogenous Nef on PBMC proliferation was associated with an increase of IFN-gamma, TNF-alpha, and IL-6 production, while, surprisingly, IL-2 production was not affected by Nef. More importantly we showed, for the first time, that Nef exerts its activating effects on PBMC proliferation through IL-15 synthesis induction by monocyte/macrophage population. In conclusion, we found that exogenous Nef protein (i) induces activation of normal PBMC, increasing their proliferative response; (ii) modulates cytokine production; (iii) exerts its activating effects through IL-15 synthesis induction; and (iv) exerts these effects entering monocyte/macrophages. Our results might suggest that Nef enhances the rate of viral replication by a novel mechanism involving the production of IL-15.

Lack of immunotoxicity of saquinavir (Ro 31-8959) used alone or in double or triple combination with AZT and ddC.

Saquinavir (Ro 31-8959; SQV) has been demonstrated to be a potent inhibitor of human immunodeficiency virus (HIV) proteinases and acts synergistically with dideoxynucleoside analogues. The aim of this study was to investigate the in vitro immunomodulatory effects of SQV on normal human peripheral blood mononuclear cells (PBMC) and on lamina propria mononuclear cells (LPMC). We used the drug either alone or in double and triple combination with AZT and ddC to assess whether SQV enhances the immunomodulatory effects induced by AZT and ddC that we previously observed. We demonstrated that SQV did not induce any modulation of the proliferative response either in PBMC or in LPMC. Similarly, NK cell-mediated cytotoxic activity and cytokine production were not modified by SQV. More importantly, SQV/AZT, SQV/ddC, and SQV/AZT/ddC combinations did not strengthen neither the inhibition of PBMC and LPMC proliferative response or the modulation of cytokine production induced by AZT, ddC, and AZT/ddC. On the other hand, the increased IL-2 production induced by AZT and ddC was not observed adding SQV to the dideoxynucleoside analogues. In conclusion, we demonstrated that SQV used in combination with AZT and ddC did not add any further immunotoxicity.

Diltiazem modulates monokine production in human mixed lymphocyte culture.

BACKGROUND: Calcium channel blockers are widely used in transplantation. Their immunosuppressive activity is well known and has been demonstrated both in vitro and in vivo. Nevertheless, their effect on cytokine production has never been reported. METHODS: One-way mixed lymphocyte cultures (MLCs) have been obtained from healthy human subjects. Cytokine production has been assessed by three different methods: by enzyme-linked immunosorbent assay on supernatants of MLC, by enzyme-linked immunospot method on MLC cells for measuring cytokine-producing cells, and by the reverse transcription polymerase chain reaction technique on MLC cells for measuring cytokine mRNAs. RESULTS: An interesting effect on proinflammatory monokines was observed: in this study, we demonstrate that the calcium antagonist diltiazem enhances interleukin-1beta and slightly reduces interleukin-6 production in MLC, but it has no effect on tumor necrosis factor-alpha levels. CONCLUSION: For the first time, a modulation of monokine production by diltiazem can be demonstrated. This evidence suggests that calcium antagonist drugs may exert effects on monocytes and possibly on other antigen-presenting cells.

Oxidized low-density lipoproteins affect natural killer cell activity by impairing cytoskeleton function and altering the cytokine network.

Several lines of evidence indicate that oxidative imbalance can play an important role in determining an impairment of natural killer (NK) cell activity in a variety of human diseases. Because a specific role for oxidized low-density lipoproteins (LDL) as pro-oxidizing agents has been envisaged, we tested the activity of oxidized LDL (ox-LDL) on NK cell-mediated cytotoxicity, cytokine release, and membrane molecule modulation. Native LDL served as control. Treatment with ox-LDL at noncytotoxic concentrations (0.2 mg/ml) during the NK/target cell (TC) interaction markedly reduced NK cytotoxic activity against U937 tumor cells. This inhibitory activity was also noticed when NK cells were pretreated with ox-LDL. Scanning electron microscopy examination of NK-target cell conjugates failed to reveal any morphological cell damage. In addition, the number of conjugates and the expression of some adhesion molecules (CD11a, CD11b, CD18, CD2, and CD62L) were not modified by ox-LDL. These observations argued against a possible interference of ox-LDL with the binding process leading to the formation of NK/TC conjugates. By contrast, immunocytochemical analyses of cytoskeleton components of NK cells exposed to ox-LDL showed a partial depolymerization and a derangement of the microtubular apparatus. These alterations were accompanied by an evident decrease in their intracellular reduced glutathione content. Owing to the important role played by the microtubular network during the killing process, it is possible to infer that a cytoskeleton alteration underlies the inhibitory activity of ox-LDL on NK cell function. In addition, exposure of mitogen-stimulated peripheral blood mononuclear cells to ox-LDL markedly reduced specific mRNA transcription and release of cytokines relevant for NK cell activity (such as tumor necrosis factor-alpha, interferon gamma, and interleukin 12). These data suggest that the impairment of NK cell activity by ox-LDL likely reflects the concomitant dysregulation of some essential mechanisms of NK cell function.

Association of HLA-DQ7 antigen with cow milk protein allergy in Italian children.

In this study we investigated the HLA association with cow milk allergy. Thirty-seven Italian children with cow milk allergy and 35 randomly selected age-matched healthy children as control group were included in the study. DNA typing was performed by restriction fragment length polymorphism (RFLP) technique. We show the first statistically significant positive association between the expression of the HLA-DQ7 antigen and cow milk allergy. Several immunological tests (skin prick test, RIA, radioallergosorbent test (RAST) and ELISA) were performed to evaluate the humoral immune responses of DQ7 positive and DQ7 negative allergic patients. Our results show that among the DQ7 positive patients the majority presented a high humoral response. Furthermore, the in vitro proliferative response of patients to the beta-lactoglobulin antigen was performed to evaluate their cell-mediated immune response. We observed that the number of the nonresponders was higher in the DQ7 positive patients when compared to the DQ7 negative patients. Our data indicate an association of HLA-DQ7 antigen with cow milk protein allergy and that the DQ7 positive patients had a prevalence of humoral rather than cellular responses.

Oxidized low density lipoproteins impair peripheral blood mononuclear cell proliferation and cytokine production.

Oxidized low density lipoproteins (ox-LDL) are known to behave as physiological pro-oxidants leading to the formation of intracellular reactive oxygen species. The presence of these altered lipoproteins in the human plasma has been associated with a number of morbid states, including atherosclerosis and immuno-deficiency. Common features of such pathological conditions seem to be represented by several alterations occurring in the immune system. In this work we analyze the in vitro effects of ox-LDL on both proliferative response and cytokine production of normal human peripheral blood mononuclear cells (PBMC). Our results indicate that ox-LDL significantly inhibit proliferative response and modulate cytokine network interfering both at protein secretion and mRNA synthesis level.

Down-regulation of interleukin-2 receptor gene activation and protein expression by dideoxynucleoside analogs.

The in vitro effect of single or combined doses of zidovudine (AZT) and dideoxycytidine (ddC) on the production and utilization of interleukin-2 (IL-2) by normal human peripheral blood mononuclear cells (PBMC) was evaluated by measuring IL-2 concentrations in supernatants from PHA-stimulated PBMC cultures. Drugs were added at the beginning of the culture period and left throughout. Whereas AZT alone (1 and 10 microM) caused only a slight increase, ddC alone (1 and 10 microM) and combined AZT/ddC (1 + 1 and 10 + 10 microM) caused a considerable increase. IL-2 gene expression in the drug-treated PBMC did not increase. This finding suggested that the increased supernatant IL-2 accumulations might be caused by a drug-induced down-regulation of the IL-2 receptor alpha (IL-2R alpha, CD25). AZT decreased IL-2R alpha expression, but only slightly. In contrast, ddC alone and combined AZT/ddC decreased the CD25 molecules in a marked and dose-dependent manner. They also markedly reduced IL-2R alpha gene expression. These findings show that the dideoxynucleoside drugs tested left PHA-induced IL-2 gene activation unchanged but decreased IL-2R alpha gene activation, thus down-regulating IL-2R alpha cell-surface protein expression.

An HIV p24 heptapeptide down-regulates antigen-specific responses in vitro interfering at the level of the T3-Ti complex.

Ch7 (RGSDIAG), a synthetic heptapeptide derived from a conserved region of HIV p24 (aa 232-238), was previously shown to suppress antigen-induced responses in cultures of normal human peripheral blood lymphocytes (PBL). We show in this paper that Ch7 is the shortest peptide retaining full inhibitory capacity. Further, the peptide inhibited efficiently and in a dose-dependent manner the induction of a specific antibody response to the antigens SRC (sheep red cells) and Candida albicans but did not exert any effect on the induction of immunoglobulin-secreting cells in PWM-stimulated cultures. Finally, Ch7 inhibited anti-CD3-induced lymphoproliferation but did not affect anti-CD2 activation. These results suggest that a conserved epitope of HIV p24 may be able to prevent the induction of antigen-specific antibody responses by interfering with lymphocyte activation via the T3-Ti complex, resulting in the abrogation of immune functions that are defective in HIV-infected individuals.

Subtelomeric as well as telomeric sequences are lost from chromosomes in proliferating B lymphocytes.

B lymphocytes purified from peripheral blood can be normally cultured in vitro for only one doubling. They can undergo an unlimited number of cell divisions after transformation with a DNA tumor virus such as the Epstein-Barr virus. We have shown that the terminal restriction fragments of virus transformed B lymphocytes are shortened in the course of proliferation and that this process is accompanied by structural modifications. We have identified the sequences that are lost during the shortening process by hybridization to the canonical human telomeric simple repeat TTAGGG, to other simple sequences that are found at the ends of human chromosomes, and to a human subtelomeric sequence. We have observed that by 20 doublings over half the TTAGGG sequences, but few or no TGAGGG sequences, are lost from the TRFs. The subtelomeric sequence was removed from most of the TRFs on which it was present. The implications that these observations have on the problems of cell senescence and oncology are discussed.

A gp120 HIV peptide with high similarity to HLA class II beta chains enhances PPD-specific and autoreactive T cell activation.

The recent report that anti-gp120 antibodies can be induced by allogeneic stimuli in experimental animals in the absence of HIV, has focused attention on the structural similarities between gp120 and MHC. Here we report that some HIV+ individuals develop antibodies which similarly react with the gp120 HIV sequence (aa 254-263) and with the HLA-DR beta chains (aa 142-151). As these two peptides share a high level of similarity, we have investigated the role of this gp120 region on HLA class II mediated T cell recognition. The synthetic peptide corresponding to the gp120 HIV sequence aa 254-263 has been tested on T cell line (TCL) activation. Both the PPD-specific and the self-HLA reactive TCL proliferation increased in the presence of this peptide. Prepulsing experiments indicate that this enhancing effect carried out by HIV peptide is exerted at the level of antigen presentation. Moreover, the specificity of this interaction is supported by the fact that a MoAb specific for this HIV peptide blocked the autoreactive TCL proliferation, similarly to the inhibition carried out by anticlass II antibody. These data support the hypothesis that the functional homology between the HIV peptide and the HLA beta chain described may be involved in the pathogenesis of AIDS.