The B-cell superantigen Finegoldia magna protein L causes pulmonary inflammation by a mechanism dependent on MyD88 but not B cells or immunoglobulins

Abstract

Objective and design: To determine whether Finegoldia magna protein L (PL) causes lung inflammation and, if so, whether the response is dependent on its immunoglobulin (Ig)-binding B-cell superantigenic property.

Material: Pulmonary inflammatory reactions were analyzed at various time points after intratracheal administration of PL to various strains of mice.

Results: PL caused peribronchial and perivascular inflammation that peaked at 18-24 h. Polymorphonuclear cells (PMNs) began to accumulate in bronchoalveolar lavage fluid (BALF) of PL-challenged mice by 4 h and accounted for >90% of leukocytes by 18-24 h. Inflammation was marked by the appearance of MIP-2, KC, TNF-α, and IL-6 in the BALF with peak levels attained 4 h after PL administration. PL-induced pulmonary inflammation was associated with increased airway hyper-reactivity following inhalation of methacholine. The inflammatory reaction was unabated in mice lacking B cells and immunoglobulins. In contrast, PL-induced inflammation was abrogated in MyD88-deficient mice. PL-induced responses required alveolar macrophages.

Conclusions: These results strongly suggest that PL-induced lung inflammation is dependent on an innate MyD88-dependent pathway rather than the Ig-binding properties of this microbial B cell superantigen. We propose that this pulmonary inflammatory reaction is caused by the interaction of PL with a Toll-like receptor expressed on alveolar macrophages.

Figure 1. PL-induced lung histopathology in C57BL/6 mice

Representative hematoxalin and eosin-stained sections of lavaged and paraffin-embedded lung tissue from C57BL/6 mice at 1–48 hours post PL challenge (n>5 at each time point) and 24 hours post BSA challenge (bottom left). PL induced peribronchial and perivascular inflammation. Original magnification, × 100; insets magnification × 400. The peak inflammatory response occurred 18–24 hours after i.t. administration of PL. Solid arrows indicate peribronchial inflammation.

Figure 1. PL-induced lung histopathology in C57BL/6 mice

Representative hematoxalin and eosin-stained sections of lavaged and paraffin-embedded lung tissue from C57BL/6 mice at 1–48 hours post PL challenge (n>5 at each time point) and 24 hours post BSA challenge (bottom left). PL induced peribronchial and perivascular inflammation. Original magnification, × 100; insets magnification × 400. The peak inflammatory response occurred 18–24 hours after i.t. administration of PL. Solid arrows indicate peribronchial inflammation.

Figure 2. Kinetics of PMN infiltration in BALF

C57BL/6 mice (n>5) were administered PL i.t. At varying time points thereafter, animals were sacrificed and their lungs were lavaged. The total number of PMNs was determined and expressed as means ± SD. PMNs began to appear in the BALF of PL-treated animals at 4 hours, peaking at 18–24 hrs.

Figure 3. BALF Cytokine/Chemokine Assay

Concentrations of MIP-2, KC, TNF, IL-6 and IL-1β were analyzed in the BALF of PL-challenged C57BL/6 mice (n>4 per group) and expressed as means ± SD. Peak levels of analytes were apparent at 4–8 hours with obvious falloff at 24 hours. MIP-2 and KC levels were significantly higher at 4 hours vs. 8 hours P= 0.010 and 0.007 (respectively); Student's t-test. Levels of each analyte were < 10 pg/ml in BSA treated animals.

Figure 4. PL-induced airway hyperreactivity

Lung function measurements were performed on a subset of PL-treated (n=6) and BSA-treated mice (n=4) 24 hours after treatment. Lung resistance was assessed using the Flexivent system in anesthetized, canulated and ventilated mice following inhalation of increasing doses of MCh as indicated. Data are expressed as the percent change over baseline. The baseline values for the PL-treated and BSA-treated groups were 0.97±0.08 and 0.96±0.09 cmH₂O.s/min. The Area Under Curve values (expressed as means ± SD) were significantly different between the PL and BSA-treated groups (p<0.05; Mann-Whitney U test).

Figure 5. PL-induced lung response in JHT and MyD88−/− mice

Eighteen hours following i.t. administration of PL to JHT (a) and MyD88−/− (c) mice and their respective wild-type controls, lungs were examined for histopathology. Shown are representative (n=3) hematoxalin and eosin stained lung sections (100× magnification) revealing the presence of an intact pulmonary inflammatory reaction in JHT mice (a) compared to similarly stained sections from PL-challenged wild-type mice (b). By contrast, histopathological abnormalities are markedly attenuated in MyD88−/− (c) vs (d) wild-type control mice. Solid arrows indicate peribronchial inflammation and stippled arrow indicates perivascular inflammation..

Figure 5. PL-induced lung response in JHT and MyD88−/− mice

Eighteen hours following i.t. administration of PL to JHT (a) and MyD88−/− (c) mice and their respective wild-type controls, lungs were examined for histopathology. Shown are representative (n=3) hematoxalin and eosin stained lung sections (100× magnification) revealing the presence of an intact pulmonary inflammatory reaction in JHT mice (a) compared to similarly stained sections from PL-challenged wild-type mice (b). By contrast, histopathological abnormalities are markedly attenuated in MyD88−/− (c) vs (d) wild-type control mice. Solid arrows indicate peribronchial inflammation and stippled arrow indicates perivascular inflammation..

Figure 6. PL-induced responses in MyD88 −/− mice

PL-treated MyD88−/− mice demonstrated a marked reduction in BALF PMN numbers vs. wild-type mice (a; n=3; P<0.005, Students t-test) and markedly reduced cytokines/chemokines in BALF vs. wild-type mice in the BALF at 4 hours (b; n=5; p<0.001, Student's t-test). Data are expressed as means ± SD.

Figure 6. PL-induced responses in MyD88 −/− mice

PL-treated MyD88−/− mice demonstrated a marked reduction in BALF PMN numbers vs. wild-type mice (a; n=3; P<0.005, Students t-test) and markedly reduced cytokines/chemokines in BALF vs. wild-type mice in the BALF at 4 hours (b; n=5; p<0.001, Student's t-test). Data are expressed as means ± SD.

Figure 7. PL-induced histopathology in C3H/Hej (TLR-4 deficient) mice)

Representative hematoxalin and eosin-stained sections of lavaged and paraffin-embedded lung tissue from TLR-4 deficient C3H/Hej mice (a) and their respective wild-type controls (b) prepared 18 hours after PL challenge. Original magnification × 100. Arrows indicate peribronchial inflammation.

Figures 8. Role of alveolar macrophages in PL-induced inflammatory reactions

C57BL/6 mice were treated i.t. with Cl2MDP-encapsulated liposomes (Lipo-clod) or Lipo-PBS as a control; PL was administered i.t. 24 hours later and BALF PMN numbers (a) and histopathological reactions (b) were then assessed 24 hours thereafter as described in the standard protocol. AM depletion by Lipo-clod decreased PL-induced BALF PMN numbers expressed as means ± SD (n=3 Lipo-PBS/PL and 4 Lipo-clod/PL-treated mice; P<0.001, Student's t-test) and histopathological reactions (representative experiment).