MCP-1 antibody treatment enhances damage and impedes repair of the alveolar epithelium in influenza pneumonitis

Abstract

Recent studies have demonstrated an essential role of alveolar macrophages during influenza virus infection. Enhanced mortalities were observed in macrophage-depleted mice and pigs after influenza virus infection, but the basis for the enhanced pathogenesis is unclear. This study revealed that blocking macrophage recruitment into the lungs in a mouse model of influenza pneumonitis resulted in enhanced alveolar epithelial damage and apoptosis, as evaluated by histopathology, immunohistochemistry, Western blot, RT-PCR, and TUNEL assays. Abrogation of macrophage recruitment was achieved by treatment with monoclonal antibody against monocyte chemoattractant protein-1 (MCP-1) after sub-lethal challenge with mouse-adapted human influenza A/Aichi/2/68 virus. Interestingly, elevated levels of hepatocyte growth factor (HGF), a mitogen for alveolar epithelium, were detected in bronchoalveolar lavage samples and in lung homogenates of control untreated and nonimmune immunoglobulin (Ig)G-treated mice after infection compared with anti-MCP-1-treated infected mice. The lungs of control animals also displayed strongly positive HGF staining in alveolar macrophages as well as alveolar epithelial cell hyperplasia. Co-culture of influenza virus-infected alveolar epithelial cells with freshly isolated alveolar macrophages induced HGF production and phagocytic activity of macrophages. Recombinant HGF added to mouse lung explants after influenza virus infection resulted in enhanced BrdU labeling of alveolar type II epithelial cells, indicating their proliferation, in contrast with anti-HGF treatment showing significantly reduced epithelial regeneration. Our data indicate that inhibition of macrophage recruitment augmented alveolar epithelial damage and apoptosis during influenza pneumonitis, and that HGF produced by macrophages in response to influenza participates in the resolution of alveolar epithelium.

Figure 1.

(A) Effect of anti–monocyte chemoattractant protein (MCP)-1 treatment on leukocyte recruitment into murine lungs after infection with 10⁴ TCID₅₀ of mouse-adapted human influenza A/Aichi/2/68 virus. Lung homogenates (LH) prepared from uninfected mice served as the uninfected control. Cellular infiltrates were evaluated in bronchoalveolar lavage fluid (BALF) samples collected on Day 4 after infection without and with treatment with anti–MCP-1 or nonimmune IgG. Total cell counts were significantly increased in virus-infected and nonimmune IgG-treated groups compared with uninfected animals. In contrast, both macrophage and neutrophil numbers were drastically diminished in the anti–MCP-1–treated group. Results are expressed as means ± SE, with n = 5 per group. *P < 0.05 versus LH group; **P < 0.05 versus infected but untreated group. (B) Changes in body weights of BALB/c mice after infection and treatment with antibodies. Animal weights were recorded daily for 4 days, and expressed as means ± SE, with n = 10 for LH and virus-infected groups, and n = 15 for anti–MCP-1 and IgG treatment groups. (C) Determination of virus titers in mouse lung homogenates. MDCK cells were infected with serial 10-fold dilutions of lung homogenates from uninfected, virus-infected, anti–MCP-1–treated, and IgG-treated animals. The numbers on the y axis represent virus titers expressed as 10^y TCID₅₀ per gram of total protein (means ± SE), with n = 5 for the virus-infected group and n = 10 for anti–MCP-1 and IgG treatment groups. The virus titers (∼ 10⁷) of the three infected groups were similar.

Figure 2.

Effect of anti–MCP-1 antibody treatment on levels of (A) serum mouse keratinocyte-derived chemokine (KC) and (B) lung MPO. Both mouse KC and myeloperoxidase (MPO) levels were significantly decreased in anti–MCP-1–treated infected animals. Data are represented as means ± SE, with n = 5 for uninfected and infected groups, and n = 10 for anti–MCP-1 and IgG treatment groups. *P < 0.05 versus LH group; **P < 0.05 versus infected but untreated group.

Figure 3.

Effect of anti–MCP-1 treatment on histopathologic changes in lungs at Day 4 after infection. Animals administered with uninfected LH displayed normal architecture of airway (BR) and alveolar epithelia (AV) without inflammation (A, B). Mice infected with influenza virus (C, D) and treated with IgG (G, H) exhibited increased cellular infiltration with mild damage of both airway and alveolar epithelia. Prominent hyperplasia of alveolar epithelial cells was also observed in both infected and IgG-treated animals (arrows). Enhanced alveolar and airway epithelial damage with enlarged airspaces and some denuded epithelial lining (arrowheads) were noted in infected animals treated with anti–MCP-1 (E, F). Tissue sections were stained with hematoxylin and eosin, and examined at magnifications of ×100 and ×400 in the left and right columns, respectively. Semiquantitative histopathology scoring was performed (n = 5 for LH and infected groups and n = 10 for anti–MCP-1 and IgG treatment groups), with the scores shown in Table 2.

Figure 4.

Evaluation of alveolar epithelial damage by Western blot, RT-PCR,and immunohistochemistry. Alveolar epithelial markers including T1-α (for type I epithelium) and surfactant protein (SP)-C (for type II epithelium) were used to evaluate alveolar epithelial damage. (A) Western blot analyses depicting T1-α and SP-C expression in lung homogenates of (1) uninfected, (2) virus-infected, (3) anti–MCP-1, and (4) IgG treatment groups. Anti–MCP-1–treated animals showed significantly reduced expression of SP-C and T1-α, with β-actin (42 kD) serving as a loading control. (B) Densitometric analyses of Western blot bands, each expressed as a percentage of the corresponding control β-actin band (with the LH group assigned as 100%). (C) Semiquantitative RT-PCR assay for mRNA expression of T1-α and SP-C. (D) Densitometric analyses of RT-PCR amplicons, each expressed as a percentage of the corresponding control β-actin amplicon (with the LH group assigned as 100%). Representative results are shown for uninfected and infected groups (n = 5 each), and for anti–MCP-1 and IgG treatment groups (n = 10 each). Asterisk denotes statistical significance at P < 0.05 versus LH group. (E) Immunostaining for detection of T1-α and SP-C in lung sections counterstained with DAPI nuclear dye. Uninfected lung section shows continuous T1-α staining (arrows) covering 95% of the alveoli. Strong SP-C signals (arrowheads) are depicted in uninfected and IgG-treated groups. However, discontinuous T1-α staining (*) and minimal SP-C staining (white arrow) were observed in anti–MCP-1–treated animals.

Figure 5.

Quantitative analysis for alveolar type II cell hyperplasia by staining lung sections with SP-C and PCNA. Out of the total SP-C–positive cells, the number and percentage of cells positive for both PCNA and SP-C were determined for each group. Results are expressed as mean percentages ± SE, with n = 5 for uninfected and infected groups, and n = 10 for anti–MCP-1 and IgG treatment groups. *P < 0.05 versus LH group; **P < 0.05 versus infected group.

Figure 6.

Detection of apoptosis by TUNEL assay. (A) Representative micrographs depicting lungs of (i) uninfected, (ii) virus-infected, (iii) anti–MCP-1 treatment, and (iv) IgG treatment groups. Arrows indicate apoptotic epithelial cells, while arrowheads show TUNEL-positive macrophages at ×400 magnification. (B) Quantification of apoptosis was determined by the number of positively stained nuclei within the alveolar epithelial lining. The apoptosis index represents the average percentage of TUNEL-positive alveolar epithelial cells, and the results are expressed as means ± SE, with n = 5 for LH and infected groups, and n = 10 for anti–MCP-1 and IgG treatment groups. *P < 0.05 versus LH group; **P < 0.05 versus infected but untreated group.

Figure 7.

Effect of anti–MCP-1 treatment on HGF production in BALF and lungs. (A) HGF levels in BALF measured by ELISA (n = 5 per group). (B) Expression of HGF mRNA in lungs analyzed by RT-PCR exemplifying HGF-specific fragments amplified from total RNAs of lungs of three representative mice per group. (C) Densitometry analysis, with each HGF RT-PCR amplicon expressed as a percentage of the corresponding β-actin control amplicon. Data are represented as means ± SE, with n = 5 for uninfected and infected groups, and n = 10 for anti–MCP-1 and IgG treatment groups. *P < 0.05 versus LH; **P < 0.05 versus infected group. (D) Immunostaining for HGF detection in lung sections and BALF cells counterstained with DAPI nuclear dye. (i) Uninfected lung section shows occasional staining in alveolar epithelial cells (arrowheads). Strong HGF signals are apparent in macrophages (arrows) of (ii) infected lung sections and (iii) BALF cells. (iv) Distinct HGF staining in macrophages (arrows) but absent in neutrophils (open arrows) in BALF.

Figure 8.

HGF expression in freshly isolated mouse alveolar macrophages incubated with influenza virus-infected LA-4 cells. (A) HGF levels in the cell culture supernatants were determined by ELISA. (B) RT-PCR analysis of HGF mRNA expression. HGF-specific fragments (upper panel) together with β-actin fragments (lower panel) were amplified from total RNAs of lungs and subjected to agarose gel electrophoresis. Lanes represent (1) macrophages, (2) LA-4 cells, (3) macrophages and uninfected LA-4 cells, (4) macrophages and LA-4 cells at 24 hours after infection, and (5) macrophages and infected LA-4 cells treated with anti–MCP-1 antibody. (C) Amplicons were subjected to densitometry, and the HGF readings expressed as percentages of the corresponding β-actin amplicons. Data are represented as means ± SE of two independent experiments. *P < 0.05 compared with cultured macrophages.

Figure 9.

Analysis of alveolar type II epithelial proliferation in murine lung explants exposed to recombinant mouse HGF or anti-HGF monoclonal antibody after influenza virus infection. (A) Immunostaining for detection of SP-C and BrdU in lung sections (at ×400 magnification) of (i) uninfected, (ii) infected, (iii) infected and recombinant HGF- treated, and (iv) infected and anti-HGF–treated lung explants. Double staining (arrows) represent alveolar type II cells undergoing proliferation. BrdU-positive cells were also observed in bronchiolar epithelium of HGF-treated lung explants (open arrow). (B) Quantification of type II epithelial cell proliferation by staining sections of lung explants with SP-C and BrdU. Out of the total SP-C–positive cells, the number and percentage of cells positive for both BrdU and SP-C were determined. Two independent experiments were performed in triplicate at each time-point, and results are expressed as mean percentages ± SE. *P < 0.05 versus uninfected.