M. Finiasz

Specific lytic activity against mycobacterial antigens is inversely correlated with the severity of tuberculosis

The ability of peripheral blood mononuclear cells (PBMC) from patients with active tuberculosis to display cytotoxic responses against autologous Mycobacterium tuberculosis (Mtb)‐pulsed macrophages was evaluated. Non‐MHC restricted cell‐dependent lytic activity was observed in ex vivo effector cells from tuberculosis patients and was mediated mainly by CD3+γδ TCR+ T (γδ T) cells bearing CD56 and/or CD16 molecules. MHC‐restricted and non‐MHC restricted cytotoxic T cells (CTL) were differentially expanded upon stimulation with Mtb in tuberculosis patients and normal controls (N). Class‐I restricted CD8+ CTL and class‐II restricted CD4+ CTL were generated in PPD+N and to a lesser extent in PPD–N. Mtb‐stimulated effector cells from tuberculosis patients became progressively non‐MHC restricted CD4–CD8–γδ T cells, while lytic activity of CD4+ and CD8+CTL decreased gradually as the disease became more severe. On the other hand, target cells were lysed by ex vivo cells from tuberculosis patients through the Fas‐FasL and perforin pathways. Mtb‐induced CD4+ CTL from tuberculosis patients and N controls preferentially employed the Fas‐FasL mechanism. Mtb‐induced CD8+ CTL effector cells from patients used the perforin‐based mechanism while cells from N controls also used the Fas‐FasL pathway. While Mtb‐induced γδ CTL from patients and PPD–N employed the latter mechanism cells from PPD+N individuals also used the perforin pathway. It can be concluded that shifts in the CTL response and the cytolytic mechanisms take place as the pulmonary involvement becomes more severe.

IL‐9 promotes anti‐Mycobacterium leprae cytotoxicity: involvement of IFNγ

Interleukin 9 (IL‐9) is a T‐cell derived factor preferentially expressed by CD4+ Th2 cells and it has been characterized both in human and murine systems. It is a pleiotropic cytokine with multiple functions on cells of the lymphoid, myeloid and mast cell lineages, as well as on lung epithelial cells. Other activities described for IL‐9 support its contribution to asthma and its important role in helminthic infections, where a Th2 response can be protective and IL‐9 enhances resistance or is responsible for elimination of the nematode. Nevertheless, until recently there were no studies on its role in bacterial infections in man. We have demonstrated that cytokines can modulate the specific cytotoxicity generation in peripheral blood mononuclear cells from leprosy patients and normal controls. In the present report we studied the effect of IL‐9 in this experimental model. Our results indicate that IL‐9 can counteract the negative effect mediated by IL‐4 on the generation of M. leprae‐induced cytotoxic T lymphocytes. Moreover, it can increase this lytic activity in controls and enhance the stimulatory effect of IL‐2 or IL‐6 in cells from leprosy patients and controls. IL‐9 is also able to revert the inhibitory effect of IL‐10 and IL‐13 on the M. leprae‐induced cytotoxic activity. Although the exact mechanism of action of IL‐9 remains to be determined, interferon gamma seems to be required for the effect of IL‐9 in this experimental model. These data suggest that IL‐9 may have an atypical Th2 behaviour and play a role in the modulation of the immune response to mycobacterial infections.

Lack of cytotoxic activity against Mycobacterium leprae 65-kD heat shock protein (hsp) in multibacillary leprosy patients

Cytotoxic T cells play an important role in host defence mechanisms, as well as in the immunopathology of leprosy. In this study, we evaluated whether Mycobacterium leprae hsp18, hsp65 and Myco. tuberculosis hsp71 could induce cytotoxic T cell activity against autologous macrophages pulsed with these hsp. Paucibacillary (PB) patients and normal controls generated more effector cells than multibacillary (MB) patients with all three hsp tested. There was no cross‐reactivity between any of the hsp tested. Mycobacterium leprae hsp65 induced cytotoxic responses only in those MB patients undergoing an erythema nodosum leprosum (ENL) episode. Although hsp65 and hsp18 induced similar proliferation in MB patients, a high proportion of these patients did not generate cytotoxic effector cells in response to hsp65. Hence, those T cells reacting to hsp65 may play an important role in the control of Myco. leprae infection.

NK cells modulate the cytotoxic activity generated by Mycobacterium leprae -hsp65 in leprosy patients: role of IL-18 and IL-13

Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL‐18 and IL‐13, two cytokines that can influence NK cell activity, in the generation of M. leprae‐derived hsp65‐cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL‐18 modulates hsp65‐induced CTL generation and collaborates with IL‐12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL‐12 cannot up‐regulate this CTL generation, while, in contrast, IL‐18 increases the cytotoxic activity both in the presence or absence of NK cells. IL‐13 down‐regulates the hsp65‐induced CTL generation and counteracts the positive effect of IL‐18. The negative effect of IL‐13 is observed in the early stages of the response, suggesting that this cytokine affects IFNγ production by NK cells. mRNA coding for IFNγ is induced by IL‐18 and reduced in the presence of IL‐13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL‐13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNγ protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL‐18 and IL‐13 and this effect takes place through the production of IFNγ.

Platelet aggregation inhibition by mononuclear leukocytes.

In this study we have investigated the effect of human mononuclear leukocytes (ML) on platelet aggregation. The results obtained demonstrated that coincubation of platelets with nonstimulated ML decreased platelet aggregation induced by collagen or thrombin in a concentration-dependent manner. The inhibitory effect increased with the incubation period of the cells, reaching a plateau at 5 minutes. T and non-T enriched ML suspensions exerted an inhibitory effect similar to the total population of ML. Supernatants from ML or mixed cell suspensions also diminished platelet aggregation. 6-keto PGF1 alpha concentration in the supernatants was less than 10 pg/ml. Hemoglobin, L-arginine and cytochrome C did not modify the antiaggregating activity of ML, whereas superoxide dismutase potentiated the inhibition of aggregation mediated by ML. The inhibitory effect was not modified by monoclonal antibody (MoAb) against the lymphocyte function-associated antigen 1, alpha subunit (LFA-1 alpha) or by a MoAb directed against P-selectin. Our results demonstrated that ML inhibited platelet aggregation, at least partially, by the release of a soluble factor(s) distinct of prostacyclin or nitric oxide. Surface adhesion molecules seem also not to be involved.

Interleukin 2 stimulates heart contractility in the presence of exogenous arachidonate or the calcium ionophore A 23187

An increase in the isometric developed tension (IDT) of isolated rat atria was observed shortly after the addition of human interleukin 2 (IL-2) to the organ preparation with subthreshold concentrations of either arachidonate (AA, 1.98 X 10(-6)M) or the calcium ionophore A 23187 (1.9 X 10(-6)M). Both natural purified IL-2 (nIL-2) and yeast recombinant IL-2 (rIL-2) were active in this experimental system. It was determined that this lymphokine was active at 2 X 10(-11)M, considering as a reference the specific activity of rIL-2. Anti-IL-2 monoclonal antibody (anti-IL-2 MAb) abolished this reaction. Inhibition of atrial phospholipase C activity by nitrocarboxyphenyl N,N-diphenylcarbamate (NCDC, 5 X 10(-6)M) prevented the development of the inotropic positive effect of IL-2 in the presence of either AA or A 23187. The synthetic diacylglyceride 1-oleoyl, 2-acetyl-glycerol (OAG) replaced the IL-2 as stimulatory signal but NCDC had no effect on the reaction. The results suggest that IL-2 can alter the physiologic behaviour of the heart and that its mechanism of action is probably similar to the one proposed for other IL-2 targets (IL-2 receptor-positive T lymphocytes, T cell lines).

Immune response to Streptococcus pneumoniae in asthma patients: comparison between stable situation and exacerbation.

In Argentina, more than 3 million people suffer from asthma, with numbers rising. When asthma patients acquire viral infections which, in turn, trigger the asthmatic response, they may develop subsequent bacterial infections, mainly by Streptococcus (S.) pneumoniae. This encapsulated Gram+ bacterium has been considered historically a T cell‐independent antigen. Nevertheless, several papers describe the role of T cells in the immune response to S. pneumoniae. We evaluated the response to S. pneumoniae and compared it to the response to Mycobacterium (M.) tuberculosis, a different type of bacterium that requires a T helper type 1 (Th1) response, in cells from atopic asthmatic children, to compare parameters for the same individual under exacerbation and in a stable situation whenever possible. We studied asthma patients and a control group of age‐matched children, evaluating cell populations, activation markers and cytokine production by flow cytometry, and cytokine concentration in serum and cell culture supernatants by enzyme‐linked immunosorbent assay (ELISA). No differences were observed in γδ T cells for the same patient in either situation, and a tendency to lower percentages of CD4+CD25hi T cells was observed under stability.

Stimulation of heart contractility by supernatants from lectin-activated lymphocytes. Role of IL-2

The positive inotropic effect of cell-free supernatants from lectin-activated lymphocytes is lost by dialysis and can be partially restored by addition of arachidonic acid (AA). Interleukin-2 (IL-2) in the presence of AA could stimulate the response of the heart while the interferons (alpha-IFN or gamma-IFN) were ineffective. The activity of PHA-L-SN and that of IL-2 + AA was neutralized by anti-IL-2 MAb. These results suggest a key role for IL-2 in the stimulation of heart contractility by supernatants from activated lymphocytes.

Human lymphocyte aggregation

The purpose of the present study was to assess lymphocyte (L) aggregation. Mononuclear cells were obtained according to Boÿum. Blood was defibrinated with glass beads and laid on Ficoll-Hypaque gradients. Monocyte depletion was achieved by adherence to plastic for 18 hs. L aggregation was assayed using the turbidimetric method. When L were challenged with collagen (1-8 micrograms/ml), ADP (2.5 microM), epinephrine (1 X 10(-4) M), ristocetin (1.5 mg/ml) and bovine F VIII no aggregation could be observed. When L were stimulated by arachidonic acid (AA) (160 microM) a complete and irreversible aggregation was obtained. This effect was markedly inhibited when L were previously incubated with aspirin (40 micrograms/ml). On the other hand 5, 8, 11, 14 eicosatetraynoic acid (ETYA), a lypoxygenase inhibitor, was not able to reverse L aggregation induced by AA. Leukotriene B4, C4 and D4 were not able to induce L aggregation. When TXA2 like material from platelets and L was transferred to L preincubated with aspirin a normal aggregation response was obtained. All these results lead to the concept that the main pathway involved in L AA metabolism is probably cyclo-oxygenase. Ionophore A 23187 was not able to induce L aggregation at any concentration employed (0.1 - 100 microM) and 6 keto PGE1 could not inhibit AA induced lymphocyte aggregation.

Production of thromboxanes by transformed lymphocytes — Effect on heart contractility

We studied the effect of transformed lymphocytes from patients with chronic lymphocytic leukaemia (CLL) and the Raji cell (Raji) on the response of rat isolated atria to sodium arachidonate (AA). In contrast to normal lymphocytes, CLL cells and Raji cells decrease the contractile tension of rat isolated atria. Addition of exogenous AA (1.98 X 10(-6) M) to Raji, further reduced the isometric developed tension. Time of culture of Raji was important, as the negative inotropic effect was greater at 72 h than at 24 h of culture. Living cells were required and cell-free supernatants were inactive. Preincubation of CLL cells or Raji with cyclooxygenase inhibitors (acetyl salycilic acid, indomethacin) or inhibitors of thromboxane (TX) synthesis (imidazole, L-8027) abolished the negative inotropic response suggesting the contribution of TXs. L-8027 also reduced the growth rate of Raji cells, indicating that TXs may play a role in the regulation of cell division. The production of TXs by CLL and Raji cells from both endogenous and exogenous sources provided additional support to this hypothesis and suggested that activation of this metabolic pathway may be related to cell transformation.