Thalidomide inhibited the synthesis of IgM and IgG whereas Thalidomide+Dexamethasone and Dexamethasone alone acted as co-stimulants with pokeweed and enhanced their synthesis.

Thalidomide (Thal) provides effective treatment for erythema nodosum leprosum (ENL). In combination with Dexamethasome (Dex) it is an effective treatment for multiple myeloma (MM) and Waldenström's macroglobulinemia (WM). Thal's mechanism(s) of action in the treatment of these diverse medical conditions is not known, but it could be suppression of immunoglobulin (Ig) synthesis. Mononuclear cells were stimulated with pokeweed (PWM), and treated with Thal, Thal+Dex or Dex. The cultures were assayed for IgM and IgG. The maximum synthesis was expected to occur in cultures stimulated with PWM at 0.5, 5.0 or 10 microg/ml. The test agents at 15 microM each were expected to alter the response. Compared to cultures stimulated with PWM alone, there was significantly less Ig in the cultures containing Thal+PWM, and significantly more Ig in the cultures containing Thal+Dex+PWM or Dex+PWM (Wilcoxon). The median % of maximum was 57 for cultures treated with Thal+PWM; 184 for cultures treated with Thal+Dex+PWM, and 139 for cultures treated with Dex+PWM. Thal also acted as a co-stimulant with PWM and enhanced the synthesis of IL-2, IL-6 and DNA; whereas, Thal+Dex or Dex enhanced Ig synthesis, but suppressed IL-2, IL-6 and cell proliferation. Thal's ability to suppress Ig may explain its activity in ENL, MM and WM. The enhancement of Ig by Dex does not help to explain a role for Dex alone or in combination with Thal for the treatment of MM and WM.

Thalidomide's ability to augment the synthesis of IL-2 in vitro in HIV-infected patients is associated with the percentage of CD4+ cells in their blood.

Thalidomide is used for treating erythema nodosum leprosum. It is also used to treat aphthous ulcers in HIV-infected patients. The mechanism of action of this drug is yet to be fully understood, but modulation of inflammatory cytokines like IL-2 and TNF-alpha may play a role. We investigated the effect of thalidomide on the production of IL-2 and TNF-alpha by staphylococcal enterotoxin A (SEA) stimulated peripheral blood mononuclear cells (PBMC) from HIV-infected patients. The PBMC from 20 patients was incubated in the presence of 4.0 microg/ml of thalidomide and 50 ng/ml of SEA. After 18 h, the culture supernatant was assayed for IL-2 and TNF-alpha. The PBMC incubated with thalidomide and SEA produced significantly more IL-2 than those incubated with SEA alone. The TNF-alpha secreted by the same cells incubated with thalidomide and SEA was not significantly different from that secreted by the cells incubated with SEA alone. The amount of IL-2 produced in the thalidomide and SEA treated cultures was directly correlated with the percentage of CD4+ cells in blood, and inversely correlated with the percentage of CD8+ cells in blood. No statistically significant correlations were found when comparing the amount of TNF-alpha produced in the thalidomide and SEA treated cultures with the percentage of CD4+ or CD8+ cells in the blood. Thalidomide can act, in vitro, as an additional stimulant to augment the synthesis of IL-2 in HIV-infected patients. Increased production of IL-2 by activated T-cells may be a mechanism through which it exerts its immunomodulatory effects.

Serum IL-1ra is elevated in lepromatous leprosy patients.

Patterns of production of specific cytokines are accepted as standards for T-lymphocyte subsets in diseases caused by intracellular parasites. These lymphocyte subsets (Th1 and Th2) have been associated with the different poles of the leprosy spectrum. Lepromatous leprosy (LL) onset correlates with cytokines produced by Th2 cells on the grounds of the patient's poor cellular immune response, i.e., interleukin 2 (IL-2) and gamma interferon (IFN-gamma) deficiency. On the other hand, tuberculoid leprosy (TL) has been associated with a Th1 response. Moreover, pro-inflammatory cytokines like IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) play a major role in chronic inflammatory pathologies being IL-1ra and TNF-alpha soluble receptors, natural counterbalancing inhibitors. In light of this background, we decided to measure serum levels of IL-1 beta, IL-1ra, TNF-alpha and IL-6 in LL and TL patients, and we also studied the production in vitro of Th1 (IFN-gamma, IL-2), Th2 (IL-4, IL-10) and TNF-alpha cytokines. Our data showed that IL-1ra is highly elevated in sera from LL patients; there were no differences in Th2 cytokine levels and there were diminished levels in Th1 cytokines.

Inhibition of apoptosis by ionomycin and zinc in peripheral blood mononuclear cells (PBMC) of leprosy patients.

PBMC from tuberculoid (BT/TT) and lepromatous leprosy (BL/LL) leprosy patients showed spontaneous apoptosis when cultured in the absence of mitogen for 24 h, which was inhibited by anti-tumour necrosis factor-alpha (TNF-alpha) antibodies. Apoptosis was also inhibited by ionomycin and zinc, which also increased IL-2 and decreased TNF-alpha production. The increase in IL-2 production suggests a mechanism whereby dietary supplements with zinc might alter the cell-mediated immunity response in leprosy patients.

Dapsone decreases the cumulative incidence of diabetes in non-obese diabetic female mice.

Dapsone (4,4'-diaminodiphenyl sulfone) has a large clinical experience due to its antimicrobial effects against Mycobacterium leprae, the causative agent of leprosy, and is used clinically where inflammation mediated by neutrophils is perceived to play a role. We administered dapsone in two concentrations (0.001% and 0.0001% w/w of diet) to 30 female non-obese diabetic (NOD) mice to explore the effect of dapsone on the development of IDDM following either a 1-week pulse or 20 weeks of continuous oral dapsone administration. Those mice receiving either the high or low doses of dapsone in the continuous group had a significantly reduced cumulative percentage of onset of IDDM. One of the seven mice given 0.0001% dapsone became diabetic (age 25 weeks), while none of the eight high dose (0.001%) mice developed the disease. Histological examination of pancreatic sections revealed islet infiltration in all groups of animals. The pulse and continuous experiments showed no statistically significant difference in the frequency or severity of lymphocytic infiltration. Dapsone administration did not inhibit growth, and growth rates were greater in those animals receiving the higher dapsone dose compared with the lower dose comparable to controls. We studied whether dapsone influenced murine lymphocyte function in addition to the published effects of the drug on neutrophils. At doses approximating those achieved in vivo (0.4 and 2 micrograms/ml), dapsone was found to inhibit murine splenocyte IL-2 and IL-4 secretion in response to concanavalin A. In view of the wide clinical experience with dapsone, randomized trials of the drug in new onset diabetes may be warranted.

Hydrolysis of thalidomide abrogates its ability to enhance mononuclear cell synthesis of IL-2 as well as its ability to suppress the synthesis of TNF-alpha.

Thalidomide is effective in the treatment of inflammatory conditions like erythema nodosum leprosum in leprosy patients, and aphthous ulcers in AIDS patients. Its mechanism of action is uncertain and reports of its effect on the synthesis of inflammatory cytokines such as IL-2 and TNF-alpha are contradictory. As thalidomide is labile to spontaneous hydrolysis at pH 7.4, studies were carried out to explore the effects of deliberate hydrolysis or the ability of thalidomide to modulated cytokine production by human mononuclear cells stimulated in vitro with Staphylococcal enterotoxin A (SEA)(IL-2) or lipopolysaccharide from Salmonella minnesota (LPS)(TNF-alpha). Unhydrolyzed thalidomide at 4.0 micrograms/ml consistently enhanced the synthesis of IL-2 in SEA-stimulated cells, and suppressed the synthesis of TNF-alpha in LPS-stimulated cells; whereas, hydrolyzed thalidomide had no enhancing effect on SEA stimulated-cell synthesis of IL-2 or suppressive effect on LPS stimulated-cell synthesis of TNF-alpha. These findings demonstrate that thalidomide's ability in vitro to enhance IL-2 and to suppress TNF-alpha in stimulated cells is dependent on the intact molecule and underscore the necessity to employ thalidomide under appropriate physicochemical conditions.

Proliferative responses of T cells from the skin and nerve lesions of leprosy patients.

In the current study we compared the mitogenic responses of T cells from skin and nerve biopsies of leprosy patients with those of peripheral blood mononuclear cells (PBMC). Lymphocytes from these sources were cultured at < or = 100 cells/well in the presence of PHA, irradiated autologous feeder cells, and IL-2, and proliferation was assessed after 6 to 12 days. Whereas PBMC were capable of vigorous responses, the growth of cells from skin and nerve was markedly reduced. The diminished response was independent of the clinical status of leprosy patients and was also observed in skin-infiltrating lymphocytes from patients suffering from other disorders. Analysis of proliferative responses at 1 cell/well suggested both a reduction in precursor frequency and a decrease in mean burst size. Analysis of lymphokine production suggested that cultured cells from skin lesions had reduced IL-w and IL-4 production relative to PBMC generated under similar conditions. Equal numbers of CD3+ cells were present in each source, but lesion cells were enriched in CD45RA- "memory" T cells, as well as CD3+CD28+ T cells. However, these alterations in subpopulation distribution could not account for the substantial differences in proliferative potential. We conclude that significant differences exist in the activation potential of cells from different tissue sources.

Does thalidomide affect IL-2 response and production?

The exact mechanism of immunosuppression by thalidomide is poorly understood. A common denominator in the pathogenesis of graft-vs.-host disease, graft rejection, reactional lepromatous leprosy, and autoimmune disorders modulated by thalidomide is the activation of T lymphocytes culminating in the synthesis of interleukin-2 (IL-2), the expression of high-affinity IL-2 receptors, and the induction of proliferation. We investigated the effect of thalidomide on the production of IL-2 by the human leukemia cell line Jurkat through induction of IL-2 gene enhancer activity and through the presence of IL-2 in supernatants. beta-galactosidase activity, encoded by a reporter lac z construct and controlled by a transcription factor in thalidomide-treated PMA- and ionomycin-stimulated Jurkat cells, was similar (97 +/- 1.33%; p > 0.1) to non-thalidomide-treated controls at all drug concentrations tested. IL-2 enhancer-driven beta-galactose activity of thalidomide-treated and stimulated cells was also similar to that of untreated controls (p > 0.2). The IL-2 production of activated nontransfected Jurkat cells was gauged by using the IL-2-dependent cell line HT-2 as a readout and by ELISA. Jurkat cells were subcloned by limiting dilution. Bulk cultures and three subclones (J.5.2.5., J.5.2.9., and J.5.3.8.) were assayed at 6, 12, and 24 hours after PHA/PMA-induced stimulation. No inhibitory effect on the IL-2 production by thalidomide could be detected at any of the drug concentrations tested (5-30 micrograms/mL), whereas 10 to 100 ng/mL of cyclosporine inhibited the IL-2 production by 95 to 100%. In addition, we observed neither inhibition of IL-2-dependent proliferation of HT-2 nor inhibition of PHA-induced proliferation of peripheral mononuclear cells by thalidomide at all drug concentrations used (5-30 micrograms/mL). These results do not support the possibility of a modulatory effect on the immune response by thalidomide via IL-2 production and IL-2 response.

IL-12 inhibits IL-4 synthesis in keyhole limpet hemocyanin-primed CD4+ T cells through an effect on antigen-presenting cells.

Although IL-12 is known to enhance IFN-gamma synthesis in unprimed CD4+ T cells, the effect of IL-12 on IL-4 synthesis in primed CD4+ T cells, which are thought to have relatively fixed cytokine profiles, has not been clearly examined. We examined the effects of IL-12 on cytokine production by CD4+ keyhole limpet hemocyanin (KLH)-primed memory lymph node T cells and by already established KLH-specific CD4+ T cell clones. First, we found that the presence of IL-12 greatly reduced the development of IL-4 synthesis in resting but not activated memory CD4+ T cells. Although IL-12 did not inhibit the production of IL-4 in cloned Th2 effector cells, it greatly inhibited the development of IL-4 synthesis in primed CD4+ T cells taken from the lymph nodes of mice previously immunized with KLH. Secondly, we found that IL-12 inhibited IL-4 synthesis either when directly added to cultures of T cells or when APC were preincubated in IL-12. Inasmuch as the enhancing effect of IL-12 on IFN-gamma synthesis occurred optimally only when the T cells were cultured directly in IL-12, these studies indicate that IL-12 affects IL-4 synthesis via a mechanism that involves APC, a process that differs from that by which it affects IFN-gamma synthesis. These studies also indicate that the administration of IL-12 would be clinically useful in treating patients, for example those with allergic disease or lepromatous leprosy, in whom memory T cells inappropriately overproduce IL-4.

In vitro effect of interleukin-2 on proliferative responses of peripheral blood T cells from leprosy patients.

Because of the important role played by interleukin-2(IL-2) in T cell growth and differentiation, we investigated the effect of exogenous IL-2 on the proliferative response of peripheral blood mononuclear cells(PBMCs) from 77 leprosy patients. The proliferative responses of PBMCs from lepromatous leprosy(LL) or borderline lepromatous leprosy(BL) patients to M. leprae were significantly lower(cpm 6,051 +/- 803 for LL type; 4,951 +/- 2,529 for BL type) than those from tuberculoid leprosy(TT) or borderline tuberculoid leprosy(BT) patients (28,853 +/- 28,916 for TT type; 15,884 +/- 334 for BT type). To investigate the effect of exogenous IL-2, purified IL-2 was added at the start of culture at 100 unit/ml. There was an apparent increase in 3H-thymidine incorporation of M. leprae-stimulated PBMCs(18,723 +/- 6,503) in the presence of IL-2 compared to the results without IL-2(6,051 +/- 803) in LL patients. Twenty nine out of 33 LL patients belonged to the responders to IL-2 and four patients were nonresponders. Therefore we conclude that the defective cell mediated immune response in LL patients may result from diminished production of IL-2, but we can not exclude the possibility of diminished expression of the IL-2 receptor. And we suggest that the immunologic heterogeneous response of an individual to M. leprae is important to the pathogenesis of clinical disease in the same LL patients.

Mitogen induced proliferation and cytokine production by lymphocytes from leprosy patients.

In this paper we have assessed the mitogen responses of leprosy patients and healthy donors in terms of proliferation and cytokine production (Interleukin 2 and Interferon gamma). The patients investigated included untreated and multidrug therapy non-responsive LL patients and MDT responsive LL and TT patients. The mitogen responses of untreated and multidrug therapy non responsive LL patients were not significantly different from those of the healthy donors. It was interesting to note that TT and multidrug therapy responsive LL patients showed higher responses to mitogens than healthy donors as assessed by all three parameters. The results suggest a correlation of increased mitogenic responses with improvement in clinical status in leprosy.

Immunologic defects in leprosy patients. II. Interleukin 1, interleukin 2, and interferon production in leprosy patients.

The capabilities of monocytes and lymphocytes in peripheral blood mononuclear leukocytes (PBML) to produce interleukin-1 (IL-1), IL-2, and interferon (IFN), respectively, were evaluated in various types and treatments of leprosy patients. IL-1 production in response to lipopolysaccharide was significantly lower in LL, BL, BB, and BT patients than in normal controls. However, there were no differences in IL-1 levels between TT patients and normal controls. The percentages of nonspecific-esterase-positive cells adhering to the plastic surfaces were not different in LL, BB and TT patients when compared to normal controls. However, they were significantly higher in BT and BL patients than in normal controls. When PBML from leprosy patients were stimulated with concanavalin-A (ConA) for IL-2 production, there were no differences in the IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients compared to normal controls. Similar results were obtained when PBML were stimulated with phytohemagglutinin-P (PHA-P). However, when purified protein derivative (PPD) was used as the stimulating agent, there were significantly lower IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients when compared to normal controls. There were also lower IL-2 levels in untreated BL/LL and BT/TT patients compared to treated BL/LL and BT/TT patients, respectively. PBML were stimulated with PHA-P or ConA for IFN production.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on lymphokine production in lepromatous leprosy patients.

In order to evaluate whether lymphokine (LK) release is impaired in patients with lepromatous leprosy (LL), the production of two LKs, namely leukocyte inhibitory factor (LIF) and interleukin-2 (IL-2) from peripheral blood mononuclear cells of LL individuals was investigated. Results show that in eight patients CD4+ cells exhibit a reduced release of LIF, while CD8+ lymphocytes are still able to secrete this LK. In the remaining three patients both CD4+ and CD8+ cells produced LIF as do normal lymphocyte subpopulations. As far as IL-2 release is concerned, all patients fail to produce the above LK either using purified CD4+ or CD8+ lymphocytes. These data emphasize additional defects in immune responsiveness in leprosy.

In vitro suppression of interleukin 2 production by Mycobacterium leprae antigen.

The suppressive activity of three different lots and sources of Mycobacterium leprae (M. leprae) was studied by measuring the inhibitory effect on interleukin 2 (IL-2) production in normal subjects. All three M. leprae preparations had suppressive activity on IL-2 production when peripheral blood mononuclear leucocytes (PBML) were stimulated with the mitogens PHA-P or Con A in a dose response. M. leprae also had suppressive activity on IL-2 production when PBML were stimulated with the specific antigen, PPD. The inhibitory activity of M. leprae on IL-2 was not due to the direct interaction of M. leprae and IL-2 because direct mixing of IL-2 with different concentrations of M. leprae did not alter the activity of IL-2. Incorporation of M. leprae for 0, 6 and 12 h in PHA-P and PBML cultures had no inhibitory effect on IL-2 production; however, after 14, 16 and 18 h of M. leprae incorporation, significant inhibitory effects were noted on IL-2 production. The suppressive mechanism of M. leprae was studied by incorporating M. leprae into PBML or adherent cells. The suppressive activity could be detected in both M. leprae-stimulated PBML and M. leprae-stimulated monocyte supernatant fluids. The suppressive mechanism of M. leprae was further evaluated by incorporating 1 and 2 micrograms/ml of indomethacin in PBML containing PHA-P and M. leprae. The suppressive activity of M. leprae was significantly diminished by indomethacin, suggesting that the inhibitory effect of M. leprae may result from the induction of PBML and adherent cells to produce the immunosuppressive activity of prostaglandin(s).