Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency

Abstract

Acute lung injury (ALI) leading to respiratory distress is a common sequela of shock/trauma, however, modeling this process in mice with a single shock or septic event is inconsistent. One explanation is that hemorrhage is often just a "priming insult," thus, secondary stimuli may be required to "trigger" ALI. To test this we carried out studies in which we assessed the capacity of hemorrhage alone or hemorrhage followed by septic challenge (CLP) to induce ALI. Lung edema, bronchoalveolar lavage interleukin (IL)-6, alveolar congestion, as well as lung IL-6, macrophage inflammatory protein (MIP)-2, and myeloperoxidase (MPO) activity were all increased in mice subjected to CLP at 24 but not 72 hours following hemorrhage. This was associated with a marked increase in the susceptibility of these mice to septic mortality. Peripheral blood neutrophils derived from 24 hours post-hemorrhage, but not Sham animals, exhibited an ex vivo decrease in apoptotic frequency and an increase in respiratory burst capacity, consistent with in vivo "priming." Subsequently, we observed that adoptive transfer of neutrophils from hemorrhaged but not sham-hemorrhage animals to neutropenic recipients reproduce ALI when subsequently septically challenged, implying that this priming was mediated by neutrophils. We also found marked general increases in lung IL-6, MIP-2, and MPO in mice deficient for toll-like receptor (TLR-4) or the combined lack of TLR-4/FasL. However, the TLR-4 defect markedly attenuated neutrophil influx into the lung while not altering the change in local cytokine/chemokine expression. Alternatively, the combined loss of FasL and TLR-4 did not inhibit the increase in MPO and exacerbated lung IL-6/MIP-2 levels even further.

Figure 1.

IL-6 (A), MIP-2 (B) and MPO (C) activity in the lung homogenates of C3H/HeN mice subjected to CLP 24 hours following hemorrhage (Hem) as compared to animals simply subjected to Hem alone/Sham-CLP, Sham-Hem/CLP, or Sham/Sham. All 72 hours post-Hem/CLP levels were markedly lower than those seen at 24 hours post-Hem. *, P < 0.05 vs. equivalent Hem alone group; #, P < 0.05 vs. 72 hours Hem before CLP group; n = 4 to 6/group.

Figure 2.

A: IL-6 levels in bronchoalveolar lavages are significantly elevated when compared to Hem/Sham-CLP, Sham-hem/CLP, or Sham-hem/Sham-CLP. B: Lung edema (Wet wt./Dry wt. [g/g]) as an index of organ injury is markedly increased in C3H/HeN mice subjected to CLP 24 hours following hemorrhage (Hem/CLP) as compared to animals simply subjected to Hem/Sham-CLP (Hem), Sham-Hem/CLP (CLP), and Sham-Hem/Sham-CLP (Sham). *, P < 0.05 vs. all other groups; n = 4/group.

Figure 3.

Hematoxylin and eosin stain of representative sections of the caudal lobe of the lung of C3H/HeN mice subjected to Sham-Hem/Sham-CLP (A), Hem/Sham-CLP (B), or Hem/CLP (C) (magnification, ×200). Note: Hemorrhage followed 24 hours later by CLP produced an increase in lung cellularity, septal thickening, and alveolar congestion (C). Such changes are less evident in the Hem/Sham-CLP mouse sections (B) and are largely absent in the Sham-Hem/Sham-CLP animals lung (A).

Figure 4.

Survival of C3H/HeN mice subjected to hemorrhage (Hem) or Sham-Hem 24 hours before CLP. *, P < 0.05 vs. Sham-Hem group; n = 18/group.

Figure 5.

While total leukocyte (WBC) number/ml blood (A) did not significantly change 24 hours post-Hem, there was a marked increase in the % of cells that were neutrophils (PMN; based on Gr1⁺ staining) (B), however, the frequency of apoptotic (A_o) PMNs markedly declined (C). *, P < 0.05 vs. all other groups; n = 4/group.

Figure 6.

Respiratory burst capacity of C3H/HeN mouse blood neutrophils isolated 24 hours post-Hem was consistently lower than that observed from Sham-Hem animals’ cells. A typical result of four independent experiments is provided.

Figure 7.

Changes in IL-6 (A), MIP-2 (B), and MPO (C) seen following adoptive transfer of peripheral blood neutrophils isolated from C3H/HeN donor mice which had been hemorrhaged (Hem) (24 hours earlier), Sham-Hem, or were untreated (normal) to neutropenic recipient, C3H/HeN mice that were subsequently subjected to CLP. Values are mean ± SE, n = 4 mice/group. *, P < 0.05 vs. equivalent group not subjected to CLP, while #, P < 0.05 vs. all other groups. ND, not detected.

Figure 8.

Hematoxylin and eosin stain of representative sections of the left lobe of the lung of recipient neutropenic mice 24 hours post-CLP (A, C) or Sham-CLP (B, D) that had received either hemorrhage (Hem; A, B) or Sham-hemorrhage (Sham-Hem; C, D) donor mouse blood neutrophils (magnification, ×200). Inset, represents the cumulative morphometric determination of % alveolar space present per field from 12 images taken per lung tissue section. Values given as mean ± SEM, n = 3/group. *, P < 0.05 vs. CLP neutropenic mouse receiving PMN from Hem mouse. #, P < 0.05 vs. Sham-CLP neutropenic mouse receiving Sham-hemorrhage mouse donor PMN.

Figure 9.

IL-6 (A), MIP-2 (B), and MPO (C) levels in the lung homogenates of C3H/HeOuJ mice hemorrhaged (Hem) 24 hours before CLP compared Hem/Sham-CLP or compared to Hem/CLP, C3H/HeJ (TLR-4 −/−) or Hem/CLP C3H/HeJ-FasL^gld (TLR-4 −/−, FasL −/−). Values given as mean ± SE, n = 5 to 6 mice/group. *, P < 0.05 vs. Hem/Sham-CLP, C3H/HeOuJ (HeOuJ), ^#, P < 0.05 vs. Hem/CLP C3H/HeOuJ or C3H/HeJ (HeJ). ^@, P < 0.05 vs. Hem/CLP C3H/HeOuJ or C3H/HeJ-FasL^gld (gld). ^‡, P < 0.05 vs. the equivalent Hem/CLP group.

Figure 10.

Hematoxylin stain of representative sections of the lung of Hem/Sham-CLP (A); Hem/CLP, C3H/HeOuJ mouse (B); Hem/CLP, C3H/HeJ (C); or Hem/CLP, C3H/HeJ-FasL^gld (D) mouse (magnification, ×400). Note: Hemorrhage followed 24 hours later by CLP produced an increase in lung cellularity, septal thickening, and alveolar congestion in all mouse strains, however, the extent of these changes was typically greater in endotoxin-sensitive (TLR+/+)/FasL+/+ mice (A, B) then either the C3H/HeJ (C) or C3H/HeJ-FasL^gld (D). Inset, represents cumulative determination of the % PMN (napthol AS-D chloroacetate esterase⁺ cells) per total number cells counted in 7 to 8 field, 25:m²; magnification, ×400. Values given as mean ± SEM, n = 3/group. *, P < 0.05 vs. Hem/Sham-CLP and ^@, P < 0.05 vs. HeOuJ:Hem/CLP or Hej-Fasl^gld:Hem/CLP.