Depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza A (H5N1) virus isolated from humans

Abstract

Previously, we observed that several virulent influenza A (H5N1) viruses which caused severe or fatal disease in humans were also lethal in BALB/c mice following dissemination of the virus to solid organs, including the brain. In contrast, one particular human H5N1 virus was nonlethal in mice and showed no evidence of systemic spread. To compare H5N1 viruses of varying pathogenicity for their ability to alter the mammalian immune system, mice were infected with either influenza A/Hong Kong/483/97 (HK/483) (lethal) or A/Hong Kong/486/97 (HK/486) (nonlethal) virus and monitored for lymphocyte depletion in the blood, lungs, and lymphoid tissue. Intranasal infection with HK/483 resulted in a significant decrease in the total number of circulating leukocytes evident as early as day 2 postinfection. Differential blood counts demonstrated up to an 80% drop in lymphocytes by day 4 postinfection. In contrast, nonlethal HK/486-infected mice displayed only a transient drop of lymphocytes during the infectious period. Analysis of lung and lymphoid tissue from HK/483-infected mice demonstrated a reduction in the number of CD4(+) and CD8(+) T cells and reduced synthesis of the cytokines interleukin-1beta and gamma interferon and the chemokine macrophage inflammatory protein compared with HK/486-infected mice. In contrast, the cytokine and chemokine levels were increased in the brains of mice infected with HK/483 but not HK/486. Evidence of apoptosis in the spleen and lung of HK/483-infected mice was detected in situ, suggesting a mechanism for lymphocyte destruction. These results suggest that destructive effects on the immune system may be one factor that contributes to the pathogenesis of H5N1 viruses in mammalian hosts.

FIG. 1

Comparison of lung virus titers (A), weight loss (B), and lethality (C) of BALB/c mice infected with 100 MID₅₀ of HK/483 (●), HK/486 (▴), or Dk/Sing (■). Four to five mice from each virus-infected group were euthanatized on the indicated days p.i., and titers of individual lungs were determined in embryonated chicken eggs. The limit of virus detection was 10^1.2 EID₅₀/ml (dotted line). The remaining seven mice from each group were observed for weight loss and mortality through a 9-day observation period.

FIG. 2

Kinetic analysis of circulating leukocytes (A) and blood differential counts (B and C) following H5N1 infection. Two to three mice were infected i.n. with 100 MID₅₀ of HK/483 (●) or HK/486 (▴) or were mock infected (⧫). An additional group was infected i.n. with 1,000 MID₅₀ of PR/8 (■) to induce a lethal infection. Total white blood cell counts were determined by microscopic counting of leukocytes in heparinized whole blood samples diluted with Turks solution. Blood smears were stained with Hema-3 differential stain, and the percentages of monocytes (Monos), polymorphonuclear neutrophils (PMNs), and lymphocytes (Lymphs) were determined in HK/483-infected mice (B) and HK/486-infected mice (C).

FIG. 3

Reduction of the cytokines IL-1β (A), IFN-γ (B), and TNF-α (C) in HK/483-infected mice. At the indicated times after infection, five individual lung, brain, and spleen tissues were removed and frozen at −70°C. Samples were thawed and homogenized in 1 ml of PBS, after which, the clarified cell lysates were assayed by ELISA. In addition, the constitutive cytokine levels (horizontal dotted-line) present in each tissue were determined by harvesting individual tissues from four uninfected 6-week-old BALB/c mice. The samples were prepared as described above, and the mean cytokine levels ± SE (error bars) for each tissue were determined. An asterisk indicates the HK/483-infected group was significantly (P < 0.05) different from the HK/486-infected group by ANOVA.

FIG. 3

Reduction of the cytokines IL-1β (A), IFN-γ (B), and TNF-α (C) in HK/483-infected mice. At the indicated times after infection, five individual lung, brain, and spleen tissues were removed and frozen at −70°C. Samples were thawed and homogenized in 1 ml of PBS, after which, the clarified cell lysates were assayed by ELISA. In addition, the constitutive cytokine levels (horizontal dotted-line) present in each tissue were determined by harvesting individual tissues from four uninfected 6-week-old BALB/c mice. The samples were prepared as described above, and the mean cytokine levels ± SE (error bars) for each tissue were determined. An asterisk indicates the HK/483-infected group was significantly (P < 0.05) different from the HK/486-infected group by ANOVA.

FIG. 3

Reduction of the cytokines IL-1β (A), IFN-γ (B), and TNF-α (C) in HK/483-infected mice. At the indicated times after infection, five individual lung, brain, and spleen tissues were removed and frozen at −70°C. Samples were thawed and homogenized in 1 ml of PBS, after which, the clarified cell lysates were assayed by ELISA. In addition, the constitutive cytokine levels (horizontal dotted-line) present in each tissue were determined by harvesting individual tissues from four uninfected 6-week-old BALB/c mice. The samples were prepared as described above, and the mean cytokine levels ± SE (error bars) for each tissue were determined. An asterisk indicates the HK/483-infected group was significantly (P < 0.05) different from the HK/486-infected group by ANOVA.

FIG. 4

Determination of the inflammatory chemokines MIP-2 (A) and MIP-1α (B) in H5-infected lung tissue. Samples were prepared as described in the legend to Fig. 3. An asterisk indicates the HK/483-infected group was significantly (P < 0.05) different from the HK/486-infected group by ANOVA.

FIG. 5

Representative light photomicrographs showing pathology, apoptosis, and viral antigen staining in the spleen. Mice were infected i.n. with 100 MID₅₀ of HK/483, and 3 days later spleen tissues were removed and processed for hematoxylin and eosin staining (A and B) and determination of TUNEL activity (C) and viral antigen expression (D). (A) Low-power photomicrograph showing lymphoid hyperplasia with formation of well-circumscribed loose-appearing secondary germinal centers at periphery of follicles (arrow). Magnification, ×50. (B) Higher-power magnification of secondary germinal centers showing pleomorphic mononuclear cells and multiple foci of nuclear condensation and fragmentation consistent with apoptosis (arrows). Magnification, ×158. (C) Same area showing abundant TUNEL-positive cells indicative of apoptosis. Magnification, ×158. (D) Same area showing immunostaining of viral antigens present in a few mononuclear cells (arrows). Magnification, ×158.

FIG. 5

Representative light photomicrographs showing pathology, apoptosis, and viral antigen staining in the spleen. Mice were infected i.n. with 100 MID₅₀ of HK/483, and 3 days later spleen tissues were removed and processed for hematoxylin and eosin staining (A and B) and determination of TUNEL activity (C) and viral antigen expression (D). (A) Low-power photomicrograph showing lymphoid hyperplasia with formation of well-circumscribed loose-appearing secondary germinal centers at periphery of follicles (arrow). Magnification, ×50. (B) Higher-power magnification of secondary germinal centers showing pleomorphic mononuclear cells and multiple foci of nuclear condensation and fragmentation consistent with apoptosis (arrows). Magnification, ×158. (C) Same area showing abundant TUNEL-positive cells indicative of apoptosis. Magnification, ×158. (D) Same area showing immunostaining of viral antigens present in a few mononuclear cells (arrows). Magnification, ×158.

FIG. 6

Increased apoptosis in lung tissue of HK/483-infected mice. Representative light photomicrographs showing in situ detection of apoptosis in lung tissue by TUNEL histology. Mice were infected i.n. with 100 MID₅₀ of HK/483 (A) or HK/486 (B), and 6 days later lung tissues were prepared for paraffin histology and developed for TUNEL activity. (A) TUNEL-positive cells can be seen primarily in association with the bronchial epithelial and subepithelial layers of HK/483-infected lung tissue. Magnification, ×158. (B) However, few or no TUNEL-positive cells were detected in HK/486-infected lung tissue.