Interferon-gamma (IFN-gamma)-dependent protection and synthesis of chemoattractants for mononuclear leucocytes caused by IL-12 in the lungs of mice infected with Cryptococcus neoformans

Abstract

We have recently demonstrated that IL-12 induced cellular inflammatory responses consisting mainly of accumulation of mononuclear leucocytes in the lungs of mice infected with Cryptococcus neoformans and protected mice against fulminant infection. We examined the involvement of endogenously synthesized IFN-gamma in such a response by investigating the effects of a neutralizing monoclonal antibody against this cytokine. The latter treatment completely abrogated the positive effects of IL-12 on survival of infected mice and prevented IL-12-induced elimination of microbials from the lungs. Histopathological examination showed that accumulation of mononuclear leucocytes in the infected lungs caused by IL-12 was clearly inhibited by anti-IFN-gamma MoAb. We also examined the local production of mononuclear cell-attracting chemokines such as monocyte chemotactic protein-1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta and IFN-gamma-inducible protein 10 (IP-10) in the lungs using a reverse transcriptase-polymerase chain reaction (RT-PCR) method. We found that these chemokines were not synthesized in the infected lungs, while IL-12 treatment markedly induced their production. Interestingly, neutralizing anti-IFN-gamma MoAb strongly suppressed IL-12-induced production of these chemokines. Similar results were obtained with MCP-1 and MIP-1alpha when their synthesis was measured at the protein level using respective ELISA kits. Our results indicate that IFN-gamma plays a central role in the protective effects of IL-12 by inducing mononuclear leucocyte-attracting chemokines and cellular inflammatory responses.

Fig. 1

Effect of neutralizing anti-IFN-γ MoAb on the protective effect of IL-12 treatment. (a) Mice received daily i.p. injections of PBS (no symbol, broken line; n = 5) or 0.1 μg of IL-12 (○; n = 5) for 7 days from the day of intratracheal instillation of 1 × 10⁵ cells of Cryptococcus neoformans. IL-12-treated mice were injected intraperitoneally with 200 μg of control rat IgG (▴; n = 5) or 200 μg of anti-IFN-γ MoAb (•; n = 5) 1 day prior to, at the day of and once a week after infection. The number of live mice was counted. NS, Not significant; *P < 0.01 compared with infected/IL-12-treated and antibody-untreated mice by the generalized Wilcoxon test. (b) Number of live microorganisms in lungs examined 3 weeks after infection in the same experiment. Bars represent the mean ± s.d. of five mice. The experiments were repeated three times with similar results. NS, Not significant; **P < 0.05 compared with infected/IL-12-treated and antibody-untreated mice by Student's t-test.

Fig. 2

(See also pp 117–119) Effect of neutralizing anti-IFN-γ MoAb on the inflammatory responses induced by IL-12. Mice received daily i.p. injections of PBS (a,e) or 0.1 μg of IL-12 (b,f) for 7 days from the day of intratracheal infection with 1 × 10⁵ cells of Cryptococcus neoformans. IL-12-treated mice were injected intraperitoneally with 200 μg of control rat IgG (c,g) or 200 μg of anti-IFN-γ MoAb (d,h) using the same schedule described in Fig. 1. Paraffin sections of the lung were prepared from mice killed 14 days after instillation of the microorganisms, stained with haematoxylin and eosin, and examined at × 40 (a,b,c,d) and × 200 (e,f,g,h) under a light microscope. Each photomicrograph represents a representative mouse (three per group). The experiments were repeated two times with similar results.

Fig. 3

Effect of neutralizing anti-IFN-γ MoAb on IL-12-induced expression of chemokine mRNA in lungs. Mice received daily i.p. injections of PBS (n = 3) or 0.1 μg of IL-12 for 7 days from the day of intratracheal infection with 1 × 10⁵ cells of Cryptococcus neoformans. IL-12-treated mice were injected intraperitoneally with 200 μg of control rat IgG (n = 3) or 200 μg of anti-IFN-γ MoAb (n = 3) using the schedule described in Fig. 1. On days 7 and 14 of infection, mice were killed and total RNA was extracted from the lungs. Subsequently, reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out for the indicated chemokines. Hypoxanthine phosphoribosyl transferase (HPRT) was used as an internal control. RNA samples were obtained separately from three mice in each group, and the results are representative of three sets of separate samples. 1, Infected/PBS-treated; 2, infected/IL-12-treated; 3, infected/IL-12 and rat IgG-treated; 4, infected/IL-12 and anti-IFN-γ MoAb-treated; M, DNA size marker.