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(Journal of Leukocyte Biology. 2000;68:180-186.)
© 2000 by Society for Leukocyte Biology

Influence of type 2 T cell responses on the severity of encephalitis associated with influenza virus infection

Masahide Kaji, Makiko Kobayashi, Richard B Pollard and Fujio Suzuki

Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas

Correspondence: Fujio Suzuki, Ph.D., Department of Internal Medicine, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0835. E-mail: fsuzuki{at}utmb.edu


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ABSTRACT
 
The role of type 2 T cell responses on the severity of post-infectious encephalitis was investigated in a mouse model of influenza virus infection. When mice were infected intracerebrally with 3.0 LD50 of A/NWS33 strain of influenza virus, they all showed clinical signs of encephalitis, and 90% of them died within 10 days of the infection. However, the post-infectious encephalitis was not demonstrated in mice exposed to 0.5 LD50 of the same virus. The mortality rates of mice infected with 0.5 LD50 of the virus were increased to levels observed in mice exposed to 3.0 LD50 of influenza virus infection, after the administration of a mixture of interleukin (IL)-4 and IL-10 (2 ng/mouse each; immediately, 1 and 2 days after the infection). In contrast, mortality rates of mice exposed to 3.0 LD50 of influenza virus were substantially decreased when these mice were treated with a mixture of monoclonal antibodies directed against IL-4 and IL-10. A predominance of type 2 T cell responses was demonstrated in splenic T cells of mice infected with 3.0 LD50 of influenza virus, although these responses were minimal in mice infected with 0.5 LD50 of the virus. After the treatment with the mixture of type 2 cytokines, an increase in the type 2 T cell responses in mice exposed to 0.5 LD50 of the virus was shown. These results indicate that type 2 T cell responses associated with the viral infection play an important role in the severity of post-infectious encephalitis induced in mice by the intracerebral infection of influenza A virus.

Key Words: interleukin-4 • interleukin-10 • influenza A virus


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INTRODUCTION
 
Each year many people are infected by influenza viruses, and although it is not life threatening for most people, it is debilitating, requiring 2–4 days of bed rest to fully recover [1 ]. Although the infection is not traumatic to most individuals, each influenza season the virus does cause substantial morbidity among school-age children and an increase in the mortality rate among the elderly [2 , 3 ]. The number of patients that have been hospitalized or lost their lives from acute pneumonia, chronic cardiopulmonary diseases, or other conditions induced by the influenza virus infection has been described [4 ]. In addition, the occurrence of many non-respiratory complications has been described in these patients infected with influenza virus [5 6 7 8 ]. One of them is the neurological complication associated with post-infectious encephalitis [5 6 7 8 ]. For example, acute disseminated encephalomyelitis and cerebellar ataxia are known to be associated with immune responses after an influenza outbreak [5 6 7 8 ]. In particular, the association of influenza and central nervous system disease has been disputed in children [9 ].

It has been shown that the balance between type 1 and type 2 T cell responses is one of the important factors for determining the severity of infectious diseases caused by certain viruses [10 11 12 ]. The type 1 T cell responses are accompanied with the production of type 1 cytokines [interferon-{gamma} (IFN-{gamma}) and interleukin (IL)-2] that have a function to generate various effector cells for cell-mediated protective immunity [13 14 15 ]. Type 1 T cell responses are actually down-regulated by type 2 cytokines (IL-4 and IL-10) released by type 2 T cells [13 14 15 ]. Type 2 T cell responses are accompanied with the increased production of type 2 cytokines, even though type 2 cytokines are known as promoters of humoral immune responses [13 14 15 ]. Also, IL-4 has been shown to be a promoter for the differentiation of naive T cells into IL-4-producing type 2 T cells [16 ]. A shift from type 1 T cell responses to type 2 T cell responses has been shown to be accompanied by the increased severity of murine AIDS induced by LP-BM5 murine leukemia virus infection [10 ] and stromal keratitis or encephalitis induced by herpes simplex virus infection [11 , 12 ]. In cases of influenza virus infection, type 1 T cell responses have been described to be enhanced during the early stage of the infection [17 ]. In response to the severity of infection, a predominance of type 2 T cell responses has been demonstrated [17 18 19 ]. However, the roles of T cell responses in the regulation of post-infectious encephalitis induced by influenza virus infection have not been determined. In the present study, the role of type 2 cytokines on the severity of post-infectious encephalitis induced in mice by the infection of neurovirulent influenza A virus was investigated. The results obtained showed that type 2 cytokines appeared after the viral infection increased the severity of post-infectious encephalitis in mice exposed to influenza virus.


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MATERIALS AND METHODS
 
Mice, cells, and viruses
Four-week-old BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were used in these experiments. Madin-Darby canine kidney (MDCK) cells were maintained in Eagle’s modified minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and antibiotics. MDCK cells, cultured in maintenance medium [EMEM supplemented with 2% FBS and 2 µg/mL of trypsin], were used for the titration of influenza virus [20 ]. Mononuclear cells (MNC) were prepared by Ficoll-Hypaque sedimentation from spleens of mice infected with or without influenza virus, as described previously [21 ]. T cells were purified from MNC using T cell enrichment columns (R & D Systems, Minneapolis, MN). The purity of these T cells was more than 96%, as described previously [21 ]. Neurovirulent influenza virus (H1N1) was obtained from the American Type Culture Collection (Rockville, MD). Before being used for the infection, the virus was propagated in the allantoic cavity of embryonated eggs, as described previously [22 ]. As a stock virus solution, allantoic cavities removed from embryonated eggs were stored at -70°C. The viral titer of the allantoic fluids was 1.7 x 108 EID50/mL (106.8 PFU/mL).

Reagents and cytokine assays
Anti-IL-4, anti-IL-10, anti-IL-2, anti-IFN-{gamma}, anti-CD3 monoclonal antibodies (mAbs), and rat IgG were purchased from PharMingen, San Diego, CA. Murine recombinant IL-2 (mrIL-2), IFN-{gamma} (mrIFN-{gamma}), IL-4 (mrIL-4), and IL-10 (mrIL-10) were obtained from PeproTech, Rocky Hill, NJ. The amount of cytokines in various specimens and culture fluids of splenic lymphocytes were determined by enzyme-linked immunosorbent assay (ELISA). The assay was performed three times, and the results were expressed as the mean of these three tests.

Infection experiments
Mice were infected intracerebrally (0.03 mL/mouse) with 0.5 or 3.0 LD50 of influenza virus. The 3.0 LD50 of influenza virus was shown when 1.5 x 103 EID/mouse of stock virus solution were injected into mice intracerebrally. A 10–20% mortality rate was consistently obtained when mice were exposed to 0.5 LD50 of the virus. The majority of mice (90% or more) died when they were exposed to 3.0 LD50 of the stock virus preparation. Mice infected with 3.0 LD50 of the virus died from encephalitis, which was exhibited by weight loss, suppression of appetite, epilepsy, and quadra paralysis. In experiments, these mice were treated with type 2 cytokines or mAbs directed against type 2 cytokines. As type 2 cytokines, mrIL-4, mrIL-10, or a mixture of these two cytokines were administered subcutaneously to mice at a dose of 2 ng/mouse once daily for 3 days beginning immediately after the infection. Doses and treatment schedules of these reagents were determined in our preliminary experiments. In additional experiments, a mixture of anti-IL-4 mAb and anti-IL-10 mAb (8 µg/mouse each) was administered to mice immediately after the influenza virus infection, and it was continued once daily for 3 days after the infection. In previous studies, this amount of the mixture of mAbs has been shown to completely decrease IL-4 and IL-10 levels in sera of mice inoculated with burn-associated type 2 T cells [23 ]. Sera of mice 6 days after thermal injury contain 190–240 pg/mL of IL-4 and 120–170 pg/mL of IL-10 [23 ]. The severity of influenza virus infection in mice treated with type 2 cytokines or their mAbs was evaluated by morbidity (mean survival time in days, MDS), mortality (survival rates), and viral growth in brains of tested groups compared with those of appropriate control groups. In this mouse model, the mortality is shown to be definitely influenced by the encephalitis caused by influenza virus infection. To determine the survival rate and MSD, mice were observed daily for 2 weeks after the viral infection. For the titration of influenza virus in organs, brains removed from three mice 3–7 days after infection with 0.5 or 3.0 LD50 of influenza virus were disrupted with a glass homogenizer (Wheaton) to make a 10% suspension in 1/100 M phosphate-buffered saline (pH 7.2) [21 ]. After centrifugation at 1,580 g for 20 min, supernatants were assayed for influenza virus in MDCK cells by a standard plaque method [20 ]. All experiments were performed twice, and the figures were expressed by mean values of the results shown by these two experiments.

Detection of type 1 and type 2 T cell responses
To detect type 1 T cell responses, IL-2-required cellular proliferations and type 1 cytokine-producing abilities of splenic T cells from mice exposed to the virus were assayed [24 ]. To induce the production of cytokines, 2 x 106 cells/mL of splenic T cells from various mice were stimulated with anti-CD3 mAb (2.5 µg/mL) or viral antigen (heat-inactivated influenza virus at 0.1 MOI for live virus) for 72 h. Inactivation procedures of influenza virus and the amount of the viral antigen used for the stimulation have been described previously [25 ]. Culture fluids harvested were assayed for IL-4, IL-10, IL-2, and IFN-{gamma} by ELISA. IL-2-required cellular proliferations of T cells were measured as follows: splenic T cells from mice exposed to influenza virus (2 x 106 cells/mL) were stimulated with anti-CD3 mAb (0.1–10 µg/mL) and cultured for 48 h in the presence of IL-2 (200 U/mL). [3H]thymidine ([3H]TdR, 0.5 µCi/well) was added to each well during the final 4 h of the cultivation, and the incorporation of [3H]TdR into these cells was measured by a liquid scintillation counter. The [3H]TdR uptake by these cells was compared with that of cells stimulated with the same mAb in the absence of IL-2. The assay was performed three times, and the results were expressed as the mean of these three tests.

Statistical analysis
The survival of mice exposed to influenza virus was analyzed by log rank test. Other data were statistically analyzed by the analysis of variance (ANOVA) followed by Fisher’s protected least-significant difference test. If a P value was lower than 0.05, the result obtained was considered significant.


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RESULTS
 
Effect of a mixture of mAbs directed against type 2 cytokines on the survival of encephalitic mice
Twenty mice infected intracerebrally with 3.0 LD50 of influenza virus were treated subcutaneously with a mixture of anti-IL-4 and anti-IL-10 mAbs (8 µg/mouse each) immediately, 1 and 2 days after the infection. Then, their survival was observed daily for 2 weeks and compared with the mortalities of control mice (20 mice) treated with rat IgG (Fig. 1 ). All of the infected mice treated with rat IgG showed encephalitis symptoms and 90% of them died within one week of the infection. However, 60% of mice exposed to the same dose of influenza virus survived when they were treated with the mAb mixture directed against IL-4 and IL-10 (8 µg/mouse each, P < 0.01 vs. control). When mice were infected with 3.0 LD50 of influenza virus, 1.2 x 104, 2.4 x 104, and 1.8 x 105 PFU/brain of influenza virus were detected in these mice 3, 5, and 7 days after infection, respectively. However, amounts of the virus in brains of these mice were markedly reduced when they were treated with the mAb mixture directed against IL-4 and IL-10 (day 3, 6.0 x 103 PFU/brain; day 5, 6.0 x 103 PFU/brain, P < 0.01; day 7, 1.6 x 103 PFU/brain, P < 0.001 vs. untreated controls). Also, post-infectious encephalitis was not observed in survived mice that were treated with the mAb mixture. These results suggest that type 2 T cell responses associated with the viral infection may play an important role in the development of encephalitis induced by influenza virus infection.



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  		Figure 1. Effect of a mixture of mAbs directed against type 2 cytokines on the survival of mice infected with influenza virus. Mice (20 mice) infected intracerebrally with 3.0 LD50 of influenza virus were treated subcutaneously with a mixture of mAbs for IL-4 and IL-10 (8 µg/mouse each) immediately, 1 and 2 days after the infection (filled circles). As a control, mice (20 mice) infected with the same amount of virus were treated with rat IgG (0.2 mL/mouse, open circles). The data shown are representative of two separate experiments.

Effect of type 2 cytokines on the morbidity and mortality of mice infected with a nonlethal dose of influenza virus
Three groups of 15 mice infected intracerebrally with 0.5 LD50 of influenza virus were treated subcutaneously with a predetermined dose of mrIL-4, mrIL-10, or both (a mixture of mrIL-4 and mrIL-10) immediately, 1 and 2 days after the infection. Another group of infected mice (20 mice) was treated with saline as a control. The results obtained are shown in Figure 2 . When infected mice were treated with type 2 cytokines at a dose of 2 ng/mouse, increased mortality rates (mrIL-4, 60%; mrIL-10, 40%) were obtained as compared with those of control mice (saline, 20%). Mean survival time in days (MSD) was shown to be 10.2 in the group treated with mrIL-4, or 10.7 in mice treated with mrIL-10 (Fig. 2) . This was compared with >12.6 MSD shown in the group of mice treated with saline. When infected mice were treated with the mixture of mrIL-4 and mrIL-10, 90% of them died within 10 days of the infection (P < 0.001 vs. control). MSD shown in this group of mice was 8.8 (P < 0.001 vs. control). When mice were infected with 0.5 LD50 of influenza virus, 100, 100, and < 20 PFU/brain of influenza virus were detected in these mice at 3, 5, and 7 days after infection, respectively. Amounts of influenza virus in brains of these mice were significantly enhanced when these mice were treated with the mixture of IL-4 and IL-10 (day 3, 2.3 x 103 PFU/brain, P < 0.01; day 5, 1.3 x 105 PFU/brain, P < 0.005; day 7, 5.2 x 105 PFU/brain, P < 0.001 vs. untreated controls). All of the infected mice treated with the IL-4/IL-10 mixture showed encephalitis symptoms. These results indicate that the severity of encephalitis induced in mice by the influenza virus infection is markedly influenced by mrIL-4 and mrIL-10 administered to the infected mice. The effects of various doses of the cytokine mixture on mortality rates of infected mice are shown in Figure 3 . The IL-4/IL-10 mixture at doses ranging from 2 to 200 ng/mouse increased mortality rates of infected mice. The maximum mortality rate was shown in the group treated with 2 ng/mouse of the IL-4/IL-10 mixture, suggesting that there is an optimum dose of the IL-4/IL-10 mixture when the mortality rate of mice exposed to 0.5 LD50 of the virus was influenced. These results indicate that certain doses of the IL-4/IL-10 mixture increase the development of influenza virus encephalitis.



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  		Figure 2. Effect of type 2 cytokines on the survival of mice infected intracerebrally with influenza virus. Mice (15 mice each) infected with 0.5 LD50 of influenza virus were treated subcutaneously with mrIL-4 (filled triangles) or mrIL-10 (filled circles), individually, or in combination (filled squares), at a dose of 2 ng/mouse immediately, 1 and 2 days after the infection. As a control, mice (20 mice) infected with the same amount of virus were treated with saline (0.2 mL/mouse, open circles). The data shown are representative of two separate experiments.



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  		Figure 3. Effect of various doses of the type 2 cytokine mixture on the survival of mice infected intracerebrally with influenza virus. Mice infected with 0.5 LD50 of influenza virus were treated subcutaneously with various doses of the mixture of mrIL-4 and mrIL-10 immediately, 1, and 2 days after infection. Percent survival was determined 14 days after influenza virus infection. The data are representative of two separate experiments, and each value shown is the mean ± SD of 10 mice.

Type 1 and type 2 T cell responses of splenic T cells from mice exposed to influenza virus
The capability of splenic T cells to produce IL-4, IL-10, IL-2, and IFN-{gamma} was examined in vitro. Splenic T cells were prepared from mice infected intracerebrally with 0.5 LD50 or 3.0 LD50 of influenza virus. Splenic T cells (2 x 106 cells/mL) from mice 4 days after the viral infection were stimulated with anti-CD3 mAb (2.5 µg/mL). Culture fluids harvested 72 h after the stimulation were assayed for IL-4, IL-10, IL-2, and IFN-{gamma} by ELISA. As shown in Figure 4A and B , significant amounts of IL-4 and IL-10 were produced by splenic T cells from mice exposed to 3.0 LD50 of influenza virus (P < 0.001 vs. control). However, splenic T cells from normal mice or mice infected with 0.5 LD50 of the virus did not produce significant amounts of these cytokines into their culture fluids. On the contrary, IL-2 and IFN-{gamma} were produced by control T cells stimulated with anti-CD3 mAb, whereas splenic T cells from encephalitis mice did not produce IL-2 and IFN-{gamma} into their culture fluids (Fig. 4C and 4D ). Furthermore, the production of IL-4 and IL-10 was markedly increased when splenic T cells were prepared from infected mice (0.5 LD50) that were treated with the mixture of mrIL-4 and mrIL-10 (2 ng/mouse each; P < 0.001 vs. control, Fig. 5 ). Furthermore, IL-4 and IL-10, but not IL-2 and IFN-{gamma}, were produced by splenic T cells from encephalitic mice (mice exposed to 3.0 LD50 of virus, mice exposed to 0.5 LD50 of virus and treated with the IL-4/IL-10 mixture) when the cytokine production of these cells was stimulated in vitro by heat-inactivated influenza virus (data not shown).



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  		Figure 4. The production of type 1 and type 2 cytokines in cultures of splenic T cells. Splenic T cells (2 x 106 cells/mL), from normal mice or mice 4 days after influenza virus infection (0.5 LD50 or 3.0 LD50, 4–5 mice/group), were stimulated with anti-CD3 mAb (2.5 µg/mL). Culture fluids harvested 72 h after the stimulation were assayed for IL-4 (A), IL-10 (B), IL-2 (C), and IFN-{gamma} (D) by ELISA. Values are expressed as the mean ± SD of three experiments.



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  		Figure 5. The production of type 1 and type 2 cytokines by splenic T cells from mice exposed to 0.5 LD50 of influenza virus and treated with the type 2 cytokine mixture. Mice infected intracerebrally with influenza virus (0.5 LD50) were treated with the mixture of mrIL-4 and mrIL-10 (2 ng/mouse, each) immediately, 1 and 4 days after infection. Five days after infection, splenic T cells (2 x 106 cells/mL) were obtained from these mice (4–5 mice/group). Then, these cells were stimulated in vitro with anti-CD3 mAb (2.5 µg/mL). Culture fluids harvested 72 h after the stimulation were assayed for IL-4 (A), IL-10 (B), IL-2 (C), and IFN-{gamma} (D) by ELISA. Values are expressed as the mean ± SD of three experiments.

It has been shown that type 1 T cells require the presence of IL-2 for their proliferations [24 , 26 , 27 ]. Therefore, IL-2-required cellular proliferations of various splenic T cells were examined in vitro. In the presence of 200 U/mL of mrIL-2, the proliferations of various splenic T cells (2 x 106 cells/mL) were stimulated with 0.1 to 10 µg/mL of anti-CD3 mAb for 48 h. As shown in Figure 6 , in response to the mAb stimulation, splenic T cells from normal mice or mice exposed to 0.5 LD50 of influenza virus proliferated. However, these cellular proliferations of splenic T cells from normal mice or mice infected with 0.5 LD50 of the virus were not demonstrated when cells were stimulated with the mAb in the absence of mrIL-2. Also, [3H]TdR was not incorporated into the mAb-stimulated splenic T cells from mice exposed to 3.0 LD50 of influenza virus or mice infected with 0.5 LD50 of virus and treated with the IL-4/IL-10 mixture, even though 200 U/mL of mrIL-2 was added to the cultures (P < 0.01 vs. values of T cells from normal mice or from mice infected with 0.5 LD50 of the virus). When T cells, prepared from normal mice, mice infected with influenza virus (0.5 LD50, 3.0 LD50), or mice infected with influenza virus and treated with IL-4/IL-10 mixture, were cultured with rmIL-2 alone, their proliferative responses were minimal and differences in their responses were not statistically significant. Also, no significance was shown on the number of splenic T cells recovered from uninfected mice, infected mice (0.5 and 3.0 LD50), or infected mice treated with the mixture of IL-4 and IL-10. These results suggest that type 1 T cell responses shown by the IL-2 requirement on the cellular proliferation were not demonstrated in cultures of splenic T cells from mice exposed to 3.0 LD50 of influenza virus or mice infected with 0.5 LD50 of the virus and treated with the IL-4/IL-10 mixture.



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  		Figure 6. IL-2 required cellular proliferations of splenic T cells. Splenic T cells (2 x 106 cells/mouse) were prepared from normal mice (open circles), mice infected with influenza virus [0.5 LD50 (open squares) or 3.0 LD50 (filled squares)] or mice infected with 0.5 LD50 of influenza virus and treated with a mixture of type 2 cytokines (immediately, 1 and 4 days after infection, filled triangles). For cellular proliferation, cells were stimulated with 0.1–10 µg/mL of anti-CD3 mAb in the presence of mrIL-2 (200 U/mL) for 48 h. [3H]TdR uptake by these splenic T cells were plotted. Dashed lines, [3H]TdR uptake by T cells from normal mice (open circles) or T cells from mice infected with 0.5 LD50 of influenza virus (open squares) after the stimulation with anti-CD3 mAb in the absence of mrIL-2. Four to five mice were used for each point shown. Results shown are representative of three separate experiments. *P < 0.01 compared with values of T cells from normal mice or mice infected with 0.5 LD50 of the virus.


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DISCUSSION
 
Various factors are involved in the host defense against influenza virus infection [28 , 29 ]. CD8+ cytotoxic T lymphocytes (CTLs) have been described as important effector cells during influenza virus infection [30 ]. The adoptive transfer of influenza virus-specific CTLs into mice exposed to lethal doses of influenza virus resulted in the reduction of the virus growth in lung tissues and a decrease in the mortality rates of these mice [31 ]. The main effector mechanism of CD8+ CTLs against influenza virus infection has been shown to be contact-dependent lysis of virus-infected cells [30 , 31 ]. CD8+ T cells have been described as a source of a variety of cytokines [32 , 33 ]. Depending on the presence of IL-12 or IL-4, CD8+ T cells are generated into IFN-{gamma}- and IL-2-producing type 1 T cells or IL-4- and IL-10-producing type 2 T cells [33 ]. Recently, it has been described that CD8+ type 1 T cells are a key cell for the virus clearance and recovery of the host from the infection [19 ]. However, the generation of influenza virus-specific type 1 T cells and the production of type 1 cytokines from these type 1 T cells are not induced effectively in hosts infected with influenza virus [34 , 35 ]. The generation of type 1 T cells and expression of type 1 T cell responses are usually suppressed by type 2 T cells and their type 2 cytokine products [13 14 15 ]. These facts suggest that the pathogenesis of influenza virus infection may be strongly influenced by type 2 T cell-associated cellular responses.

A predominance of type 2 T cell responses has been shown to be induced in hosts by various conditions [36 37 38 39 40 41 ]. Doses and varieties of antigens [36 37 38 ], routes of antigen delivery [39 ], and expression of specific costimulatory molecules [39 40 41 ] are suggested as factors in the generation of type 2 T cells. In our preliminary studies, a 0.5 LD50 dose of influenza virus was shown to be the highest non-lethal dose in mice (less than 5–10% mortality rates). On the other hand, a 3.0 LD50 dose of this virus was shown to be the lowest lethal dose (more than 95% mortality rates). Mice exposed to 3.0 LD50 of influenza virus exhibited encephalitis shown by weight loss, suppression of appetite, epilepsy, and quadra paralysis. However, mice infected with 0.5 LD50 of the virus did not show any symptoms of encephalitis. Therefore, we used the doses of 3 LD50/0.5 LD50 of influenza virus in these series of experiments. The severity of encephalitis and mortality rates of mice infected intracerebrally with nonlethal doses of influenza virus was markedly increased after the administration of the mixture of mrIL-4 and mrIL-10 at doses ranging from 2 to 20 ng/mouse. In contrast, the morbidity and mortality of encephalitic mice (mice exposed to 3.0 LD50 of influenza virus) were decreased when they were treated with the mixture of mAbs directed against type 2 cytokines (a mixture of anti-IL-4 mAb and anti-IL-10 mAb). IL-2 required cellular proliferations stimulated with anti-CD3 mAb were not demonstrated in cultures of splenic T cells from mice exposed to 3.0 LD50 of influenza virus or from mice infected with 0.5 LD50 of the virus and treated with the IL-4/IL-10 mixture. After the stimulation with anti-CD3 mAb or heat-inactivated influenza virus, splenic T cells from encephalitis mice produced IL-4 and IL-10 into their culture fluids. However, splenic T cells from encephalitic mice did not produce IL-2 and IFN-{gamma} after the same stimulation. Furthermore, viral growth in brains of mice exposed to 0.5 LD50 of influenza virus increased when these mice were treated with the IL-4/IL-10 mixture. These results indicate that different types of T cell responses may be expressed in mice exposed to various doses of influenza virus, and type 2 T cell responses play an important role on the pathogenesis of post-infectious encephalitis induced in mice by an intracerebral infection of influenza virus.

In the case of herpes virus encephalitis (HSE), the pathogenic role of type 2 T cells or their type 2 cytokine products has been well described [12 ]. The severity of HSE was markedly increased in HSE mice treated with type 2 cytokines or inoculated with type 2 T cells [12 ]. The contribution of type 1/type 2 T cell responses on the development of encephalitis induced in mice by infection with Theiler’s virus has also been demonstrated [27 , 42 , 43 ]. CD4+ T cells from susceptible SJL/J mice exposed to the virus showed characteristics for type 2 T cells [42 , 43 ]. However, these type 2 T cells were not demonstrated in resistant C57BL/6 mice at any time after the infection [44 ]. These facts indicate the key role of type 2 T cell responses on the development of viral encephalitis. In the present study, the post-infectious encephalitis induced by the intracerebral infection of influenza virus was shown to be type 2 T cell response-associated encephalitis. A predominance of type 2 T cell responses induced by the viral infection may cause enhanced viral growth in brains because the generation of type 1 T cells, which are essential cells against influenza virus infection, are inhibited by type 2 cytokines [13 14 15 ].

Previous studies have described that severe encephalitis is often observed in patients with influenza virus infection [9 , 45 , 46 ]. This risk is especially hazardous for children [9 , 45 , 46 ]. From this study, it is suggested that encephalitis associated with influenza virus infection may be controlled, in part, through the regulation of type 2 T cell responses associated with viral infection. If so, an inhibitor of type 2 T cells or type 2 cytokines may have a protective effect against post-infectious encephalitis induced by influenza virus infection. Soluble IL-4 receptor (sIL-4R), one of the typical inhibitors of IL-4, has been shown to decrease the morbidity and mortality of mice infected with Leishmania major or Candida albicans through the regulation of type 2 T cell responses [47 , 48 ]. We have recently demonstrated similar effects of sIL-4R against HSV-1 infection in thermally injured mice [49 ]. The susceptibility of thermally injured mice to infection with HSV-1 was shown to be 100 times greater than that of normal mice [50 , 51 ]. CD8+CD11b+ TCR{gamma}/{delta}+ IL-4- and IL-10-producing T cells were identified as cells responsible for the increased susceptibility of thermally injured mice to the infection, as the susceptibility of normal mice inoculated with burn-associated type 2 T cells to HSV-1 infection increased to levels observed in thermally injured mice [50 , 51 ]. The resistance of thermally injured mice exposed to HSV-1 was recovered when sIL-4R was administered. These facts suggest a possibility that the severity of post-infectious encephalitis induced by influenza virus infection may be regulatable immunologically by using the inhibitor of type 2 T cell responses. On the other hand, there are some descriptions that the development of type 1 T cell responses or the generation of CTLs causes the increased severity of neurological diseases [52 , 53 ]. Multiple sclerosis and acute disseminated encephalitis are described as type 1 T cell-associated neurological diseases [54 ]. In these cases, the inhibition of type 2 T cell responses may enhance the severity of encephalitis. To explore details for the pathogenesis of influenza-associated encephalitis, further experiments are needed.

Received November 1, 1999; revised March 31, 2000; accepted April 10, 2000.


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