Delta 9-tetrahydrocannabinol treatment suppresses immunity and early IFN-gamma, IL-12, and IL-12 receptor beta 2 responses to Legionella pneumophila infection

The marijuana cannabinoid, delta 9-tetrahydrocannabinol (THC), suppresses immunity to Legionella pneumophila and development of Th1 activity and cell-mediated immunity. In the current study, THC effects on cytokines regulating the development of Th1 cells were examined. BALB/c mice showed significant increases in serum IL-12 and IFN-gamma within hours of infection; however, the levels of these Th1-promoting cytokines as well as resistance to a challenge infection were suppressed by THC (8 mg/kg) injected 18 h before priming. The Th2-promoting cytokine, IL-4, was increased within hours of a Legionella infection and was further increased by THC treatment. These results suggested that THC injection suppressed the cytokine environment promoting Th1 immunity. In additional experiments, THC pretreatment and infection of IL-4 knockout mice showed that serum IL-12 and IFN-gamma were suppressed equally in both knockout and normal mice. This suggested that the drug-induced increase in IL-4 was not responsible for the decreases in serum IL-12 and IFN-gamma. However, THC treatment was shown to suppress the expression of IL-12 receptor beta 2 mRNA, indicating that, in addition to suppression of IL-12, THC injection suppressed the expression of IL-12 receptors. Finally, the role of cannabinoid receptors in Th1-promoting cytokine suppression was examined, and results with receptor antagonists showed that both cannabinoid receptors 1 and 2 were involved. It is suggested that suppression of Th1 immunity to Legionella is not due to an increase in IL-4 production but to a decrease in IFN-gamma and IL-12. Furthermore, both types of cannabinoid receptors are involved.     

Leishmania: naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors.

Russo, D. M., Chakrabarti, P., and Higgins, A. Y. 1999. Leishmania: Naive human T cells sensitized with promastigote antigen and IL-12 develop into potent Th1 and CD8(+) cytotoxic effectors. Experimental Parasitology 93, 161-170. The differentiation of naive human T cells into Leishmania-specific Th1 or cytotoxic effector cells was examined by sensitizing T cells in vitro with dead Leishmania antigen in the presence or absence of IFN-gamma or IL-12. These Leishmania-specific T cell lines proliferated and produced cytokines in response to challenge with autologous Leishmania-infected macrophages. Sensitization in the presence of IL-12 or IFN-gamma induced Leishmania-specific human Th1 responses, with IL-12 inducing more potent Th1 responses. However, IL-12-induced Th1 responses were IFN-gamma dependent. T cell lines exhibited Th2 or Th0 phenotypes when primed in the absence of cytokines. Only T cell lines primed in the presence of IL-12 contained high percentages of CD8(+) cells. These cells lysed autologous Leishmania-infected but not uninfected macrophages in an MHC-dependent manner. Thus, this in vitro sensitization system can be used to delineate the conditions for optimally priming human Leishmania-specific effector cells.     

Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines

Virulence in Toxoplasma gondii is strongly influenced by the genotype of the parasite. Type I strains uniformly cause rapid death in mice regardless of the host genotype or the challenge dose. In contrast, the outcome of infections with type II strains is highly dependent on the challenge dose and the genotype of the host. To understand the basis of acute virulence in toxoplasmosis, we compared low and high doses of the RH strain (type I) and the ME49/PTG strain (type II) of T. gondii in outbred mice. Differences in virulence were reflected in only modestly different growth rates in vivo, and both strains disseminated widely to different tissues. The key difference in the virulent RH strain was the ability to reach high tissue burdens rapidly following a low dose challenge. Lethal infections caused by type I (RH) or type II (PTG) strain infections were accompanied by extremely elevated levels of Th1 cytokines in the serum, including IFN-gamma, TNF-alpha, IL-12, and IL-18. Extensive liver damage and lymphoid degeneration accompanied the elevated levels of cytokines produced during lethal infection. Increased time of survival following lethal infection with the RH strain was provided by neutralization of IL-18, but not TNF-alpha or IFN-gamma. Nonlethal infections with a low dose of type II PTG strain parasites were characterized by a modest induction of Th1 cytokines that led to control of infection and minimal damage to host tissues. Our findings establish that overstimulation of immune responses that are normally necessary for protection is an important feature of acute toxoplasmosis.     

A single intradermal administration of soluble leishmanial antigen and plasmid expressing interleukin-12 protects BALB/c mice from Leishmania major infection.

In murine leishmaniasis, the induction of the T-helper type 1 (Th1) response contributes to infection resistance, whereas the establishment of the Th2 response makes the mice susceptible to infection. Interleukin-12 (IL-12) plays a pivotal role in the diversification of immune responses to the Th1 type. In this study, we tested whether the co-administration of IL-12 expression plasmid which compose p35 and p40 subunits and soluble leishmanial antigen (SLA) will skew the susceptible BALB/c mice to Th1 response and protect from leishmaniasis. When the mice were intradermally injected with the combination of IL-12 plasmid and SLA 7 days prior to the challenge with 1x10(6) promastigotes of Leishmania major, the local lesions completely healed and the parasite burden in the local lymph nodes significantly decreased. The cured mice attained long-term immunity, and were resistant to any subsequent rechallenge of the lethal dose of the parasite. The protective effect was associated with the development of a Th1 response, as demonstrated by the enhanced level of antigen-specific interferon-gamma (IFN-gamma) and dominant production of IgG2a in the serum. In contrast, the administration of empty plasmid plus SLA or IL-12 plasmid alone failed to protect the disease and shape the Th1 response. Furthermore, the protective efficiency induced by the vaccination was clearly prevented by the injection of either neutralizing anti-IL-12 mAb or anti-IFN-gamma mAb. The IL-12 expression plasmid is thus an effective adjuvant for the elicitation of a protective Th1 response against leishmaniasis and is therefore, considered to be appropriate for vaccinations that require the induction of Th1 type immunity.     

A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. We therefore investigated whether such a CpG-containing ODN (CpG ODN) given mucosally in the female genital tract could enhance innate immunity and protect against genital herpes infection. Groups of C57BL/6 mice were treated intravaginally with either CpG ODN or a non-CpG ODN control in the absence of any antigen either 2 days before or 4 h after an intravaginal challenge with a normally lethal dose of herpes simplex virus type 2 (HSV-2). Mice treated with CpG ODN exhibited significantly decreased titers of HSV-2 in their vaginal fluids compared with non-CpG ODN-treated mice. Furthermore, CpG ODN pretreatment significantly protected against development of disease and death compared to non-CpG ODN pretreatment. Most strikingly, CpG ODN conferred protection against disease and death even when given after the viral challenge. The CpG ODN-induced protection was associated with a rapid production of gamma interferon (IFN-gamma), interleukin-12 (IL-12), IL-18, and RANTES in the genital tract mucosa following CpG ODN treatment. The observed protection appeared to be dependent on IFN-gamma, IL-12, IL-18, and T cells, as CpG ODN pretreatment did not confer any significant protection in mice deficient in IFN-gamma, IL-12, IL-18, or T cells. Further, a complete protective immunity to reinfection was elicited in CpG ODN-treated, HSV-2-challenged mice, suggesting a role for mucosally administered CpG ODN in inducing the development of an acquired immune response in addition to its potent stimulation of innate immunity.     

IL-12 induces monocyte IL-18 binding protein expression via IFN-gamma

IL-18 is a Th1 cytokine that synergizes with IL-12 and IL-2 in the stimulation of lymphocyte IFN-gamma production. IL-18 binding protein (IL-18BP) is a recently discovered inhibitor of IL-18 that is distinct from the IL-1 and IL-18 receptor families. In this report we show that IL-18BPa, the IL-18BP isoform with the highest affinity for IL-18, was strongly induced by IL-12 in human PBMC. Other Th1 cytokines, including IFN-gamma, IL-2, IL-15, and IL-18, were also capable of augmenting IL-18BPa expression. In contrast, IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma-inducible protein-10, and Th2 cytokines such as IL-4 and IL-10 did not induce IL-18BPa. Although monocytes were found to be the primary source of IL-18BPa, the induction of IL-18BPa by IL-12 was mediated through IFN-gamma derived predominantly from NK cells. IL-18BPa production was observed in cancer patients receiving recombinant human IL-12 and correlated with the magnitude of IFN-gamma production. The IFN-gamma/IL-18BPa negative feedback loop identified in this study may be capable of broadly controlling immune activation by cytokines that synergize with IL-18 to induce IFN-gamma and probably plays a key role in the modulation of both innate and adaptive immunity.     

IL-12p40 and IL-18 modulate inflammatory and immune responses to respiratory syncytial virus infection

Respiratory syncytial virus-induced bronchiolitis has been linked to the development of allergy and atopic asthma. IL-12 and possibly IL-18 are central mediators orchestrating Th1 and/or Th2 immune responses to infection. To determine a possible role for IL-12 in regulating the immune response to acute respiratory syncytial virus infection, IL-12p40 gene-targeted (IL-12p40-/-) and wild-type mice were intratracheally infected with respiratory syncytial virus, and lung inflammatory and immune responses were assessed. Lung inflammation and mucus production were increased in the airways of IL-12p40-/- mice as compared with those of wild-type mice, concurrent with increased levels of the Th2 effector cytokines IL-5 and IL-13. Respiratory syncytial virus clearance and levels of Th1 effector cytokine IFN-gamma were not altered. Interestingly, IL-18, another mediator of IFN-gamma production, was significantly increased in the lungs of IL-12p40-/- mice early during the course of infection. Abrogation of IL-18-mediated signaling in IL-12p40-/- mice further enhanced Th2 immune response and mucus production in the airways during respiratory syncytial virus infection but failed to modulate IFN-gamma production or viral clearance. These findings implicate a role for IL-12 and IL-18 in modulating respiratory syncytial virus-induced airway inflammation distinct from that of viral clearance.     

Virus-specific memory and effector T lymphocytes exhibit different cytokine responses to antigens during experimental murine respiratory syncytial virus infection.

Mice sensitized to the G (attachment) or F (fusion) glycoproteins of respiratory syncytial virus (RSV) expressed different patterns of cytokine production and lung pathology when challenged by intranasal infection with RSV. Five days after challenge, mice sensitized to G glycoprotein produced high levels of interleukin-4 (IL-4) and IL-5 in the lungs and spleens and developed extensive pulmonary eosinophilia, while mice sensitized to F glycoprotein produced IL-2 and developed a mononuclear cell infiltration. Memory lymphocytes isolated 2 weeks after intranasal challenge of mice primed to the G or F glycoprotein secreted only IL-2 and gamma interferon (IFN-gamma) when stimulated with RSV. IL-4 and IL-5 production characteristic of Th2-type effectors in the lung was observed only after multiple rounds of in vitro stimulation of RSV G-specific memory T lymphocytes with antigen. Also IFN-gamma production appeared to play only a minor role in the expression of pulmonary pathology characteristic of Th1 or Th2 T-lymphocyte responses, because mice genetically deficient in IFN-gamma production by gene disruption displayed the same pattern of pulmonary inflammation to RSV infection after priming to RSV F or G as conventional mice. These results suggest that effector T lymphocytes exhibit a different pattern of cytokine production than memory T-lymphocyte precursors precommitted to a Th1 or Th2 pattern of differentiation. Furthermore, these observations raise the possibility that the cytokine response of human memory T lymphocytes after a single exposure to antigen in vitro may not accurately reflect the cytokine response of differentiated effector T lymphocytes at the site of infection in vivo.     

Repeated intratracheal challenge with particulate antigen modulates murine lung cytokines

When lungs of experimental animals are repeatedly challenged with Ag, pulmonary inflammation wanes via unknown mechanisms. We hypothesized that changes in the balance of lung cytokines are responsible for immune down-regulation to repeated Ag challenge. We used intratracheal (IT) challenge of primed C57BL/6 mice with SRBC and on various days after single (1IT) or triple (3IT) challenge counted lung inflammatory cells and measured whole-lung cytokine mRNA and protein concentrations using RT-PCR and ELISA. We found that lung lymphocyte numbers and parenchymal lung inflammation decreased significantly at days 6 and 9 after final Ag challenge in 3IT mice compared with 1IT mice. Lungs of 3IT mice showed the following changes in relative mRNA expression: an earlier peak in IL-10, decreased IL-1beta, and a change from a Th2 response in 1IT mice to a Th1 response in 3IT mice (with pronounced increases in IL-12, IL-18, and IFN-gamma and decreased IL-4, IL-13, and IL-5). Similar types of changes were seen in whole-lung protein concentrations for TNF-alpha, IL-10, IL-12 p40, IFN-gamma, and IL-4. Additionally, mRNA expression of the endothelial selectins CD62E and CD62P decreased and lung lymphocyte apoptosis increased in the 3IT group. Thus, physiologic down-regulation of the pulmonary immune response to repeated Ag exposure is characterized by increased anti- and decreased proinflammatory cytokines that accompanies Th1 polarization. Similar mechanisms may act to minimize chronic lung inflammation in the majority of normal humans who do not develop progressive lung pathology when repeatedly exposed to inhaled or aspirated environmental Ags.     

Production of cytokines by peritoneal macrophages and splenocytes after exposures of mice to low doses of X-rays

We have shown previously that irradiations of mice with 0.1 or 0.2 Gy of X-rays stimulate anti-tumour cytotoxic activities of peritoneal macrophages and splenocytes enriched for NK lymphocytes and suppress the development of pulmonary tumour colonies. The up-regulated cytotoxicities were related to the production of nitric oxide by macrophages, and perforin and Fas ligand by the splenocytes, but specific blockade of these pathways did not totally suppress the effector cell-mediated cytolysis of the tumour target. Hence, other factors such as cytotoxic/cytostatic cytokines might have been produced by the effector cells. To test this possibility peritoneal macrophages and splenocytes were isolated from BALB/c mice which had been either once or tentimes whole body-irradiated with the total doses of 0.1 and 0.2 Gy of X-rays and assayed for the levels of IL-1beta, IL-2, IL-12, IFN-gamma and TNF-alpha in the incubation medium using the respective ELISA kits. The results demonstrate that both single and multiple exposures to the two low doses of X-rays significantly stimulate secretion of IL-1beta, TNF-alpha and IL-12 by macrophages and IL-2 and IFN-gamma by splenocytes, but the kinetics and magnitude of the induced changes in the production of these cytokines differ between the two irradiation protocols.     

The role of IL-12 in maintaining resistance to Leishmania major

IL-12p40 is required for the maintenance of resistance during Leishmania major infection. In this study, we addressed how IL-12 mediates this function. First, we demonstrated that both subunits of IL-12, p40 and p35, were required for continued resistance to L. major. Second, using IL-12, IL-4 doubly deficient mice, we investigated the possibility that IL-12 inhibits IL-4-induced outgrowth of Th2 cells that might compete with Th1 cells. We found that even in the absence of a Th2 response, IL-12 was still required to maintain resistance. Next, using adoptive transfer of Thy-1 disparate CD4(+) T cells from L. major-healed mice, we were able to show that the loss of a protective response in L. major-infected IL-12-deficient mice is linked with the loss of Th1 cells. In contrast, there was an equal recovery of CD4(+) Th1 cells from wild-type and IL-12-deficient mice when transferred into mice that were not challenged with L. major. The ability of Th1 cells to survive regardless of IL-12 levels in the absence of Ag stimulation was confirmed by adoptive transfer studies of CD4(+) Th1 cells from DO11.10 TCR transgenic mice. Taken together, these results indicate that, rather than modulating Th2 responses or optimizing IFN-gamma production, the critical role for IL-12 in maintaining cell-mediated immunity may be to prevent the loss of Th1 cells during a challenge infection.     

Role of Th1 and Th2 cytokines in BCG-induced IFN-gamma production: cytokine promotion and simulation of BCG effect

Induction of a T-helper-type 1 (Th1) immune response is indispensable for successful treatment of superficial bladder cancer with BCG. In this study possible involvement of various cytokines in BCG action as well as their potential roles in enhancing and mimicking BCG effect were explored. In immunocompetent cell cultures, IFN-gamma, a major Th1 cytokine, appears to be a late responsive cytokine to BCG stimulation. Its induction requires involvement of various endogenously produced Th1 and Th2 cytokines. Functional abolishment of any one of these cytokines (IL-2, IL-6, IL-12, IL-18, GMCSF, TNF-alpha, or IFN-alpha, except IL-10) by neutralizing antibodies leads to reduced IFN-gamma production (19-82% inhibition in mouse and 44-77% inhibition in human systems, respectively). In mice cytokines IL-2, IL-12, IL-18, and GMCSF are observed to synergize with BCG for IFN-gamma production, whereas in human cytokines IL-2, IL-12, TNF-alpha, and IFN-alpha exhibit similar synergistic effects. Rational combinations of these Th1-stimulating cytokines (IL-12 plus IL-18 in mice and IL-2 plus IL-12 in humans, respectively) dramatically up-regulate IFN-gamma production that is incomparably superior to BCG for induction of this cytokine. These results suggest that combined Th1-stimulating cytokines and combinations of BCG plus selected Th1-stimulating cytokines are rational candidates for further study in the treatment of bladder cancer patients.     

IL-4 and IL-10 antagonize IL-12-mediated protection against acute vaccinia virus infection with a limited role of IFN-gamma and nitric oxide synthetase 2

Resistance or susceptibility to most infectious diseases is strongly determined by the balance of type 1 vs type 2 cytokines produced during infection. However, for viruses, this scheme may be applicable only to infections with some cytopathic viruses, where IFN-gamma is considered as mandatory for host defense with little if any participation of type 2 responses. We studied the role of signature Th1 (IL-12, IFN-gamma) and Th2 (IL-4, IL-10) cytokines for immune responses against vaccinia virus (VV). IL-12-/- mice were far more susceptible than IFN-gamma-/- mice, and primary CTL responses against VV were absent in IL-12-/- mice but remained intact in IFN-gamma-/- mice. Both CD4+ and CD8+ T cells from IL-12-/- mice were unimpaired in IFN-gamma production, although CD4+ T cells showed elevated Th2 cytokine responses. Virus replication was impaired in IL-4-/- mice and, even more strikingly, in IL-10-/- mice, which both produced elevated levels of the proinflammatory cytokines IL-1alpha and IL-6. Thus, IL-4 produced by Th2 cells and IL-10 produced by Th2 cells and probably also by macrophages counteract efficient anti-viral host defense. Surprisingly, NO production, which is considered as a major type 1 effector pathway inhibited by type 2 cytokines, appears to play a limited role against VV, because NO sythetase 2-deficient mice did not show increased viral replication. Thus, our results identify a new role for IL-12 in defense beyond the induction of IFN-gamma and show that IL-4 and IL-10 modulate host protective responses to VV.     

Lipoprotein I, a TLR2/4 ligand modulates Th2-driven allergic immune responses

Asthma is an inflammatory lung disease that is initiated and directed by Th2 and inhibited by Th1 cytokines. Microbial infections have been shown to prevent allergic responses by inducing the secretion of the Th1 cytokines IL-12 and IFN-gamma. In this study, we examined whether administration of lipoprotein I (OprI) from Pseudomonas aeruginosa could prevent the inflammatory and physiological manifestations of asthma in a murine model of OVA-induced allergic asthma. OprI triggered dendritic cells to make IL-12 and TNF-alpha, with subsequent IFN-gamma production from T cells. OprI stimulation of dendritic cells involved both TLR2 and TLR4. Intranasal coadministration of OprI with OVA allergen resulted in a significant decrease in airway eosinophilia and Th2 (IL-4 and IL-13) cytokines and this effect was sustained after repeated allergen challenge. The immediate suppressive effect of OprI (within 2 days of administration) was accompanied by an increase in Th1 cytokine IFN-gamma production and a significant, but transient infiltration of neutrophils. OprI did not redirect the immune system toward a Th1 response since no increased activation of locally recruited Th1 cells could be observed upon repeated challenge with allergen. Our data show for the first time that a bacterial lipoprotein can modulate allergen-specific Th2 effector cells in an allergic response in vivo for a prolonged period via stimulation of the TLR2/4 signaling pathway.     

The effects of gamma irradiation on the survival and development of Clonorchis sinensis metacercariae

The effects of gamma irradiation on the survival and development of C. sinensis metacercariae were studied to evaluate the feasibility of irradiation as a control measure for clonorchiasis. Pseudorasbora parva were collected at an endemic river of clonorchiasis and were used for irradiation of the fluke in three schemes. The first (Scheme 1) was irradiation of the isolated metacercariae from the fish followed by infection to experimental rats. The second (Scheme 2) was irradiation of the fish, and then the metacercariae were isolated and infected to rats. The third (Scheme 3) was irradiation on the rat livers after infection with normal metacercariae. Irradiation doses varied from 5 to 100 Gy for Schemes 1 and 2, and 10 to 25 Gy for Scheme 3. The rats were sacrificed 2 to 6 weeks after infection. In Scheme 1, the metacercariae irradiated at 50 Gy failed to survive in the rats after 2 or 6 weeks. However, 1 to 44% of the metacercariae irradiated at 5-30 Gy survived. The estimated LD50 of Scheme 1 was 16.5 Gy. The flukes irradiated in Scheme 2 survived better than those in Scheme 1. The average worm recovery rate in 50 Gy was 28%(7-39% individually). Increasing the dose up to 100 Gy brought a remarkably low survival rate of an average 1%(0-3% individually). The LD50 of Scheme 2 was 47.5 Gy. Worm recovery rates in the 10 Gy group of Scheme 3 were 21-39%, and those in the 25 Gy group were 2% and 34%. Although the metacercariae were irradiated, all of the recovered worms were morphologically normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lasting effects of an impairment of Th1-like immune response in γ-irradiated mice: A resemblance between irradiated mice and aged mice

Although one of the several chronic effects of ionizing radiation is aging, there are no experimental data on radiation-induced immunological aging. The most interesting change in aging was a helper T (Th) 1/Th2 imbalance. We investigated chronic effect on immune responses after ionizing radiation and its effects in irradiated mice were compared with those of aged mice. The 2-month-old mice received a whole-body irradiation of 5 Gy. At 6 months after irradiation, we compared the immune functions of the irradiated mice with those of normal mice of the same age and with those of older. Interferon (IFN)-γ and antigen-specific immunoglobulin (Ig)G2a level were lower in the irradiated mice than in normal mice of same age, showing similar levels to those of old normal mice. In contrast, interleukin (IL)-4 and IL-5 and antigen-specific IgG1 level were increased in irradiated mice when compared with the same aged-normal mice. Next, we investigated the low expression of IL-12p70, IL-12 receptors and IL-18 receptors in irradiated and old mice. Also, the decrease of natural killer cell activity was intensified in the irradiated mice, showing lower than values to those of old mice. Interestingly, in irradiated mice, the absolute numbers and the percentages of natural killer (NK) cells was extremely decreased. But the absolute numbers of Th cells and cytotoxic T (Tc) cells in old mice were significantly decreased. In conclusion, an immunological imbalance by the whole-body irradiation of 5 Gy induces to persist in the long term, resulting in the similar results with aging. Our results suggest that the downregulation of the Th1-like immune response shown in old mice rapidly occurred through exposure of ionizing radiation.