Th1-cytokine induction and anti-tumor effect of 55 kDa protein isolated from Aeginetia indica L., a parasitic plant

 We have isolated a 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by affinity chromatography on an N-hydroxysuccinimide-activated Sepharose High Performance column bound with F3, a monoclonal antibody that neutralizes the cytokine-inducing and anti-tumor effect of AIL. In the present study, we examined this protein (AILb-A) for cytokine induction and anti-tumor effects by animal study, using syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. AILb-A administration resulted in markedly increased levels of Th1 cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-12 and IL-18] in the sera derived from Meth-A-bearing mice. The in vitro re-stimulation with AILb-A of splenocytes derived from AILb-A-primed mice also selectively induced Th1-type cytokines and antigen-specific killer cell activity. The neutralizing test using cytokine-specific antibodies revealed that AILb-A-induced IL-18 plays a most significant role for IFN-γ- and killer cell-inducing activities. Furthermore, IL-12 and IL-18 induced by AILb-A inhibited specifically IL-10 and IL-4 production, respectively. Finally, we examined the anti-tumor effect of AILb-A in both Meth-A-bearing BALB/c mice and Meth-A-bearing nude mice with BALB/c background. AILb-A exhibited a striking anti-tumor effect in normal BALB/c mice inoculated with Meth-A cells. In athymic nude mice, the anti-tumor effect of AILb-A was relatively weak. These findings strongly suggested that AILb-A is a potent Th1 inducer and may be a useful immunotherapeutic agent for patients with malignant diseases. Received: 27 July 2000 / Accepted: 13 March 2001     

Activation of human peripheral blood-derived monocytes by OK-432 (Streptococcus pyogenes): augmented cytotoxicity and secretion of TNF and synergy with rIFN-gamma

The biological response modifier OK-432 has been shown both to exert the enhancement of several immunological activities and to have a direct anti-tumor effect. The present study examines the immunopotentiating effect of OK-432 on peripheral blood monocytes (PBM) derived from normal humans. Monocyte activation was assessed by examining direct cell-mediated cytotoxic activity (CMC) and secretion of cytotoxic factors in the supernatant by the 51Cr release assay and secretion of tumor necrosis factor (TNF)-alpha detected by a sensitive radioimmunoassay. The OK-432-augmented activity was compared to that achieved by recombinant interferon-gamma (rIFN-gamma). Coculture of PBM with OK-432 overnight resulted in significant augmentation of CMC and secretion of cytotoxic factors and TNF in the supernatant. The effects observed were dose dependent and the resulting activity was much more pronounced than that achieved with an optimal concentration of IFN-gamma. The monocyte- and supernatant-mediated cytotoxic activities were in a large part attributed to TNF as both activities were inhibited by anti-TNF antibody. Several parameters of monocyte activation by OK-432 were examined. The kinetics of monocyte activation revealed that a short time exposure (2-6 hr) was sufficient for activation but maximal activation was detected after 18 hr. However, the kinetics of the cytotoxic assay were not shortened and 16-20 hr was necessary for optimal cytotoxic activity. Significant synergy was obtained when suboptimal concentrations of OK-432 and IFN-gamma were used. The synergy was noted in CMC, supernatant activity, and TNF concentration. These results demonstrate that OK-432 is a potent activator of monocyte cytotoxicity and also activates secretion of TNF. Also, OK-432 is a much more potent activator than rIFN-gamma. The synergy with OK-432 and IFN-gamma suggests that OK-432-mediated activation of monocytes takes place by a different mechanism than that mediated by rIFN-gamma. Thus, monocytes and products thereof may actively participate in the in vivo anti-tumor effect mediated by OK-432.     

In situ expression of interleukin-10 and interleukin-12 in active human cutaneous leishmaniasis

Abstract Th1-type cellular immune responses (interferon-γ) play a critical role in protection against Leishmania spp. infection, whereas Th2-type cytokines (interleukin (IL)-4, IL-10) have a counter-protective effect. IL-12, a potent inducer of Th1-type cellular immune responses, may play a pivotal role in the development of a protective response. We found that IL-10 and IL-12 mRNAs were expressed in most lesions of individuals with active cutaneous leishmaniasis. The quantity of IL-12 mRNA was highly variable but correlated strongly with the level of interferon-γ expression. IL-12 expression also paralleled the expression of IL-10, a potent in vitro suppressor of IL-12 and interferon-γ production. The more chronic, non-healing lesions generally had higher levels of IL-12 mRNA indicating that the expression of this cytokine alone was not sufficient to induce healing. Although the in situ production of IL-10 did not appear to block IL-12 expression, IL-10 may still promote disease by direct suppression of macrophage activation.     

Antitumor effect induced by a hot water extract of Chlorella vulgaris (CE): Resistance to meth-A tumor growth mediated by CE-induced polymorphonuclear leukocytes

Summary When a hot water extract of Chlorella vulgaris (CE) was injected into the peritoneal cavity of BALB/c mice inoculated with syngeneic Meth-A tumor cells, the survival times were strikingly prolonged. Furthermore, peritoneal exudate cells (PEC) rich in polymorphonuclear cells (PMN) obtained from normal mice 24 h after CE injection exhibited an antitumor effect in a Winn-type assay using normal recipients. Such an activity of PEC remained almost intact after T cell or macrophage depletion. However, such PEC did not express an antitumor effect in a Winn-type assay using irradiated recipients. It was suggested that CE-induced PEC, presumably PMN, expressed an antitumor effect in cooperation with a host- or recipient-derived element(s) sensitive to irradiation. The anti-tumor mechanism of CE may be different from that of OK-432, one of the biological response modifiers.     

Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions.

Spontaneous abortion is the most common complication of pregnancy, but the etiology of a significant proportion of abortions is still unknown. We have examined the production of Th1- and Th2-type cytokines by women with unexplained recurrent spontaneous abortion (RSA) since it appears that successful murine pregnancy occurs in a Th2-dominant situation and that Th1-type immunity is associated with pregnancy failure. We have compared maternal reactivity toward placental antigens in women with a history of successful pregnancy with that in women with a history of RSA. This was done by coculturing maternal peripheral blood mononuclear cells (PBMC) with autologous placental cells and also by stimulating maternal PBMC with antigens from a choriocarcinoma cell line of trophoblastic origin. We detected significantly greater levels of the Th2 cytokines IL-6 and IL-10 in normal pregnancy compared to unexplained RSA and significantly higher levels of the Th1 cytokine IFN-gamma in RSA compared to normal pregnancy. These results suggest that women with normal pregnancy have a higher Th2 bias, while women with a history of RSA evince a bias toward Th1-type reactivity.     

Streptococcal preparation OK432 promotes functional maturation of human monocyte-derived dendritic cells

Streptococcal preparation OK432 is an immunomodulatory agent extensively used as adjuvant therapy for gastric cancer in Japan. OK432 augments the cytotoxic activity of various effector cells such as lymphocytes, macrophages and (natural killer) NK cells and induces the production of multiple cytokines. Dendritic cells (DC) are professional antigen-presenting cells (APC) that can be used for cancer vaccine therapy. In the present study, we investigated the effect of OK432 on the activation of DC. Here we report that OK432 induced phenotypic and functional maturation of human monocyte-derived DC. In vitro culture of immature DC generated from adherent peripheral blood mononuclear cells (PBMC) using granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) with OK432 at various doses (0.01 to 0.1 KE/ml) for 2 days resulted in increased cell surface expression of CD80, CD83, CD86 and ICAM-1 in a dose-dependent manner. The expression of CD83, a selective marker of mature DC, on DC activated by OK432 (OK-DC) was maximally enhanced after 3 days of incubation. Assay of cytokine production in OK-DC after 2 days in culture revealed that OK432 was a strong inducer of IL-12 and interferon-gamma (IFN-gamma). OK432 efficiently augmented the primary allogeneic T-cell responses by DC. This distinct phenotypic profile and allostimulatory capacity of OK-DC was stable for at least 48 h of additional culture in the absence of any cytokines. Moreover, the antiviral cytotoxic T lymphocytes (CTL) response in vitro was also enhanced by the addition of OK432 to the cultures. These findings suggest that OK432 is a potent stimulator of DC, and that stimulated DC are strong inducers of the T helper 1 (Th1)-type response. We conclude that OK-DC are likely candidates for use as an adjuvant for DC-based cancer immunotherapy.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.     

Systemic administration of IL-18 promotes diabetes development in young nonobese diabetic mice

IL-18 is now identified as a pleiotropic cytokine that acts as a cofactor for both Th1 and Th2 cell development. Type 1 diabetes is considered a Th1-type autoimmune disease, and to date, the suppressive effect of exogenous IL-18 on the development of diabetes has been reported in 10-wk-old nonobese diabetic (NOD) mice. In the present study we administered exogenous IL-18 systemically in 4-wk-old NOD mice using i.m. injection of the IL-18 expression plasmid DNA (pCAGGS-IL-18) with electroporation. Contrary to previous reports, the incidence of diabetes development was significantly increased in NOD mice injected with pCAGGS-IL-18 compared with that in control mice. Systemic and pancreatic cytokine profiles deviated to a Th1-dominant state, and the the frequency of glutamic acid decarboxylase-reactive IFN-gamma-producing CD4(+) cells was also high in the IL-18 group. Moreover, it was suggested that the promoting effect of IL-18 might be associated with increased peripheral IL-12, CD86, and pancreatic IFN-inducible protein-10 mRNA expression levels. In conclusion, we demonstrate here that IL-18 plays a promoting role as an enhancer of Th1-type immune responses in diabetes development early in the spontaneous disease process, which may contribute to elucidating the pathogenesis of type 1 diabetes.     

Therapeutic efficacy of sequential therapy with OK-432, cyclophosphamide,IL2-cultured lymphocytes andin vivo IL2 against advanced murine plasmacytoma

BALB/c mice inoculated IP with a syngeneic plasmacytoma MOPC104E were treated with a combination of a streptococcal preparation, OK-432 (1 KE, 0.1 mg/mouse), low-dose of cyclophosphamide (CPA, 1 mg/kg) and adoptive transfer of tumor-bearer-spleen cells (2 x 10(7) cells) cultured with IL2 and sonicated tumor extract (adoptive immunotherapy; AIT). The consecutive protocol of OK-432 (day 8, 9 post inoculation) - CPA (day 10) - AIT (day 11) was the most effective. Rate of complete remission was highest when recombinant (r-) IL2 was injected to the mice after AIT. Moreover, another bacterial preparation, Nocardia rubra cell wall skeleton and another low-dose chemotherapy, Mitomycin C could be used successfully instead of OK-432 or CPA. Transfer test of intraperitoneal cells (tumor cells plus host cells) of mice on day 11 post inoculation (on the day of AIT) revealed that OK-432 augmented the susceptibility of peritoneal cells to cultured lymphocytes in inhibition of transplantability, and that CPA after OK-432 augmented the anti-tumor effect of tumor-bearer-spleen cells which act synergistically with cultured lymphocytes. This therapy schedule seems to be the best model to augment the effect of AIT with minimal side effect.     

Thymus and activation-regulated chemokine (TARC/CCL17) produced by mouse epidermal Langerhans cells is upregulated by TNF-alpha and IL-4 and downregulated by IFN-gamma

Thymus and activation-regulated chemokine (TARC/CCL17) is a Th2-type chemokine and its receptor CC chemokine receptor 4 (CCR4) is preferentially expressed on Th2 cells. Langerhans cells (LC) are immature dendritic cells (DC) in the epidermis of the skin and play vital roles in immune response. In this study, we investigated TARC expression by murine freshly isolated LC and 48 h cultured (mature) LC, and the regulation of TARC production in cultured LC by various cytokines. Murine LC was prepared using a panning method from BALB/c mice. RT-PCR was performed using fresh and cultured LC to evaluate TARC mRNA levels. ELISA was carried out using supernatant of cultured LC to calculate secreted TARC protein levels. TARC mRNA was strongly upregulated during maturation of murine LC. TARC production by murine LC was upregulated by TNF-alpha and IL-4 and downregulated by IFN-gamma, dose-dependently. Th1 and Th2 cytokines reciprocally regulate the production of Th2-type chemokine TARC by murine LC. Th2 cytokine microenvironments in skin may increase TARC production by mature LC, providing attraction of Th2 cells in skin. This may be an amplification circuit in Th2-dominant inflammatory skin disease like atopic dermatitis.     

IL-4 induces differentiation and expansion of Th2 cytokine-producing eosinophils

Innate effector cells that produce Th2-type cytokines are critical in Th2 cell-mediated immune responses. However, it is not known how these cells acquire the ability to produce Th2 cytokines. IL-4 is a potent inducer that directs differentiation of naive CD4(+) T cells into CD4(+) Th2 effector cells. To determine whether IL-4 can induce differentiation and expansion of Th2 cytokine-producing innate cells, we used mice whose il-4 gene was replaced by a knock-in green fluorescence protein (gfp) gene. We found that, directly ex vivo, IL-4 increased the number of GFP(+) cells in the airway and the lung tissue in an Ag-specific manner. The majority of GFP(+) cells were eosinophils, suggesting that IL-4 plays a pivotal role in expanding IL-4-producing eosinophils in vivo. IL-4-producing eosinophils showed some unique features compared with IL-4-producing CD4(+) T cells. They exhibited biallelic expression of the il-4 gene when stimulated and were more dominant IL-4- and IL-5-producing cells. Furthermore, we show that IL-4 drove bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils in vitro. These results strongly suggest IL-4 is a potent factor in directing bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils.     

Streptococcal preparation OK-432: a new maturation factor of monocyte-derived dendritic cells for clinical use

For vaccinations based on dendritic cells (DCs), maturation of DCs is critical to the induction of T-cell responses. We tested the efficacy of streptococcal preparation OK-432 as a Good Manufacturing Practice (GMP)-grade maturation agent. OK-432 is currently used in Japan as a cancer immunotherapy drug. Immature monocyte-derived dendritic cells (imMo-DCs) isolated from human peripheral blood monocytes stimulated with granulocyte-macrophage colony stimulating factor and interleukin-4 were exposed to maturation factors, i.e., lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha) plus prostaglandin E2 (PGE2), and OK-432 for 2 days. OK-432 increased expression of activation- and maturation-related molecules such as HLA-DR, CD80, CD83, and CD86 in imMo-DCs at levels similar to that of TNF-alpha plus PGE2, and higher than that of LPS. All agents examined induced allogeneic T-cell proliferation at a similar level. Only OK-432 caused significant production of interleukin-12 (IL-12) p70 and interferon gamma (IFN-gamma) at both the mRNA and protein levels in imMo-DCs. Neutralizing antibody against IL-12 p70 blocked IFN-gamma secretion from OK-432-stimulated Mo-DCs. IL-12 p70 produced by OK-432-stimulated imMo-DCs induced secretion of IFN-gamma by CD4+ T cells. OK-432 and LPS activated nuclear factor kappa B (NF-kappaB) in imMo-DCs. Both secretion of IL-12 p70 and IFN-gamma and activation of NF-kappaB induced by OK-432 were suppressed when imMo-DCs were pretreated with cytochalasin B. These results indicate that uptake of OK-432 by imMo-DCs is an early critical event for IL-12 p70 production and that NF-kappaB activation induced by OK-432 also contributes partially to IL-12 p70 production. In conclusion, OK-432 is a GMP-grade maturation agent and may be a potential tool for DC-based vaccine therapies.     

Pronounced antitumor effect of LAK-like cells induced in the peritoneal cavity of mice after intraperitoneal injection of OK-432, a killed Streptococcal preparation

Summary More than 80% of BALB/c mice bearing BAMC-1 ascites tumor were completely cured after five consecutive (once every 2 days) i. p. injections of a 0.1 mg dose of OK-432, beginning on day 2 after tumor implantation. The antitumor effect of OK-432 was abolished in athymic nu/nu mice and in anti-thymocyte globulin-treated euthymic BALB/c mice, so although OK-432 treatment did increase the length of survival, all animals eventually died as a result of tumor growth. When peritoneal exudate cells (PEC), obtained on day 12 from OK-432-treated BAMC-1-bearing euthymic mice were evaluated for in vivo tumor neutralization activity, all mice receiving an i. p. injection of the admixture of the nonadherent PEC (1×10⁷ cells) with BAMC-1 cells (1×10⁵) survived for more than 60 days. When the same nonadherent PEC (1×10⁷ cells) were i. p. transferred adoptively 1 day after the inoculation of 1×10⁵ BAMC-1 tumor cells, again all mice survived.When these in vivo active PEC were tested for cytotoxicity in vitro against fresh BAMC-1 tumor cells, natural killer (NK) sensitive syngeneic RL 1, NK-sensitive allogeneic YAC-1 cells, NK-resistant syngeneic Meth-A cells, allogeneic tumor cells (EL4, B16, and P815) and xenogenic human cells, the PEC were found to be capable of lysing BAMC-1 tumor cells together with almost all of the other tumor cells, including NK-resistant cells. Nonadherent PEC contained at least two subpopulations of killer cells. One, directed to syngeneic BAMC-1 cells, was both Thy1.2 and asialo GM1 positive, and another, directed to allogeneic YAC-1 cells, was asialo GM1 positive but Thy1.2 negative. A cold target inhibition assay also suggested the presence of more than two subpopulations.These results indicate that T cells play a determined role in the immunotherapeutic effect of OK-432 on BALB/c mice bearing BAMC-1 tumor, although the participation of activated macrophages could not be excluded. The cells responsible for killing BAMC-1 and other tumor cells appearing in the PEC on day 12 were characterized as containing at least two kinds of lymphokine-activated killer cells.     

Inhibition of in vitro LAK generation by OK-432

Summary The present study was designed to investigate the in vitro effect of OK-432 on interleukin-2-(IL-2) induced lymphokine activated killer (LAK) generation, and especially to test whether OK-432 can substitute for IL-2 or act in synergism with IL-2 for activation of cytotoxic lymphocytes. Surprisingly, our results showed that the addition of OK-432 to 4-day LAK activation cultures significantly inhibited both the generation of cytotoxic effectors to the natural killer (NK) resistant Daudi cell line and the proliferative responses of lymphocytes in a dose dependent manner. The inhibition of activation was total at 0.5 KE/ml of OK-432, a dose which was still effective in augmenting NK activity against K562. The addition of penicillin G potassium (PCGk), which is contained in OK-432 at a concentration of 134,700 units/mg of dried cocci, to the LAK culture system also inhibited LAK generation at equivalent concentrations as contained in the OK-432 preparation. This inhibition of LAK generation by OK-432 was significantly eliminated by dialysis of OK-432. These results indicated that the inhibition of LAK generation was partly due to PCGk contained in the OK-432 preparation, and that OK-432 did not act synergistically with IL-2 in standard LAK activation systems.     

Human metapneumovirus induces a profile of lung cytokines distinct from that of respiratory syncytial virus

Lung cytokine and chemokine production by BALB/c mice infected with human metapneumovirus (hMPV) was compared to respiratory syncytial virus (RSV)-infected mice. hMPV infection induced lower levels of the inflammatory cytokines interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha but was a more potent inducer of granulocyte-macrophage colony-stimulating factor and triggered a more sustained production of the CXC chemokine KC compared to RSV. hMPV was a stronger inducer of both alpha interferon (IFN-alpha) and IFN-gamma responses than RSV. In regard to immunomodulatory cytokines, hMPV failed to induce detectable IL-10 or IL-12p70 but was a potent inducer of IL-12 p40 subunit. The implications for hMPV pathogenesis are discussed.     

Schistosoma mansoni and alpha-galactosylceramide: prophylactic effect of Th1 Immune suppression in a mouse model of Graves' hyperthyroidism

Graves' hyperthyroidism, an organ-specific autoimmune disease mediated by stimulatory thyrotropin receptor (TSHR) autoantibodies, has been considered a Th2-dominant disease. However, recent data with mouse Graves' models are conflicting. For example, we recently demonstrated that injection of BALB/c mice with adenovirus coding the TSHR induced Graves' hyperthyroidism characterized by mixed Th1 and Th2 immune responses against the TSHR, and that transient coexpression of the Th2 cytokine IL-4 by adenovirus skewed Ag-specific immune response toward Th2 and suppressed disease induction. To gain further insight into the relationship between immune polarization and Graves' disease, we evaluated the effect of Th2 immune polarization by helminth Schistosoma mansoni infection and alpha-galactosylceramide (alpha-GalCer), both known to bias the systemic immune response to Th2, on Graves' disease. S. mansoni infection first induced mixed Th1 and Th2 immune responses to soluble worm Ags, followed by a Th2 response to soluble egg Ags. Prior infection with S. mansoni suppressed the Th1-type anti-TSHR immune response, as demonstrated by impaired Ag-specific IFN-gamma secretion of splenocytes and decreased titers of IgG2a subclass anti-TSHR Abs, and also prevented disease development. Similarly, alpha-GalCer suppressed Ag-specific splenocyte secretion of IFN-gamma and prevented disease induction. However, once the anti-TSHR immune response was fully induced, S. mansoni or alpha-GalCer was ineffective in curing disease. These data support the Th1 theory in Graves' disease and indicate that suppression of the Th1-type immune response at the time of Ag priming may be crucial for inhibiting the pathogenic anti-TSHR immune response.     

In Vivo Modulation of Vaccine-Induced Immune Responses toward a Th1 Phenotype Increases Potency and Vaccine Effectiveness in a Herpes Simplex Virus Type 2 Mouse Model

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4⁺-T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.     

Elevated levels of interleukin-13 and IL-18 in patients with dengue hemorrhagic fever

Interleukin (IL)-13 is produced by T helper 2 (Th2)-type cells and inhibits the production of proinflammatory cytokines by activated monocytes, while IL-18 is a pleiotropic cytokine that induces interferon-gamma and plays an important role in the development of Th1-type cells. Role of the shift from a Th1-type response to Th2-type has been suggested in the pathogenesis of dengue hemorrhagic fever (DHF). This study was undertaken to investigate the possible protective/pathogenic role of IL-13 and IL-18 in patients with DHF. Sera were collected from a total of 84 patients with various grades of dengue illness and 21 normal healthy controls and tested for IL-13 and IL-18 levels using commercial enzyme-linked immunosorbent assay kits. The results showed that very low levels of IL-13 (4+/-3 pg ml(-1)) and IL-18 (15+/-4 pg ml(-1)) were detected in the sera of healthy controls. In dengue patients, the levels of IL-13 and IL-18 were the highest in the patients with DHF grade IV (205+/-103 pg ml(-1) and 366+/-155 pg ml(-1), respectively) and the lowest in patients with dengue fever (22+/-12 pg ml(-1) and 76+/-50 pg ml(-1), respectively). Both the cytokines appeared (IL-13=20+/-11 pg ml(-1) and IL-18=70+/-45 pg ml(-1)) during the first 4 days of illness and reached peak levels (IL-13=204+/-96 pg ml(-1) and IL-18=360+/-148 pg ml(-1)) by day 9 onwards. The presence of high levels of IL-13 and IL-18 during severe illness and late phases of the disease suggests that both of these cytokines may contribute to the shift from a Th1- to Th2-type response and thus to the pathogenesis of DHF.