Pulmonary contusion primes systemic innate immunity responses

Abstract

Introduction: Traumatic injury may result in an exaggerated response to subsequent immune stimuli such as nosocomial infection. This "second hit" phenomenon and molecular mechanism(s) of immune priming by traumatic lung injury, specifically, pulmonary contusion, remain unknown. We used an animal model of pulmonary contusion to determine whether the injury resulted in priming of the innate immune response and to test the hypothesis that resuscitation fluids could attenuate the primed response to a second hit.

Methods: Male, 8 to 9 weeks, C57/BL6 mice with a pulmonary contusion were challenged by a second hit of intratracheal administration of the Toll-like receptor 4 agonist, lipopolysaccharide (LPS, 50 microg) 24 hours after injury (injury + LPS). Other experimental groups were injury + vehicle or LPS alone. A separate group was injured and resuscitated by 4 cc/kg of hypertonic saline (HTS) or Lactated Ringer's (LR) resuscitation before LPS challenge. Mice were killed 4 hours after LPS challenge and blood, bronchoalveolar lavage, and tissue were isolated and analyzed. Data were analyzed using one-way analysis of variance with Bonferroni multiple comparison posttest for significant differences (*p < or = 0.05).

Results: Injury + LPS showed immune priming observed by lung injury histology and increased bronchoalveolar lavage neutrophilia, lung myeloperoxidase and serum IL-6, CXCL1, and MIP-2 levels when compared with injury + vehicle or LPS alone. After injury, resuscitation with HTS, but not Lactated Ringer's was more effective in attenuating the primed response to a second hit.

Conclusion: Pulmonary contusion primes innate immunity for an exaggerated response to a second hit with the Toll-like receptor 4 agonist, LPS. We observed synergistic increases in inflammatory mediator expression in the blood and a more severe lung injury in injured animals challenged with LPS. This priming effect was reduced when HTS was used to resuscitate the animal after lung contusion.

Figure 1. Lung histology shows that pulmonary contusion primes innate immunity for enhanced neutrophil response

We compared lung histology of animals given LPS with no antecedent trauma (LPS alone), animals undergoing blunt chest trauma followed by intratracheal instillation of LPS (Injury+LPS) and injured animals administered intratracheal saline (Injury+vehicle). Second hit animals (Injury+LPS) show an increased pulmonary neutrophilic infiltrate and alveolar septal edema when compared to animals given LPS alone. Hemorrhage and structural distortion is evident in injured animals. Data shown (100× magnification) is representative of histology for at least 3 animals in each group.

Figure 2. Pulmonary contusion primes innate immunity for enhanced neutrophil response

We compared the neutrophil response of animals given LPS with no antecedent trauma (LPS alone) to animals given a blunt chest trauma followed by IT instillation of LPS (Injury+LPS) or injured animals administered IT saline (Injury+vehicle). Second hit animals (Injury+LPS) have increased BAL neutrophilia (A) and lung MPO activity (B) when compared to animals given LPS alone or injured animals administered IT saline (Injury+vehicle). Data shown is the mean ± SEM. Differences are significant (p≤0.01) between all groups (n=5 for each group).

Figure 3. Pulmonary contusion primes the systemic innate immune response

We compared the serum levels of IL-6, CXCL1 and MIP-2 in animals given LPS with no antecedent trauma (LPS alone) to animals given a blunt chest trauma followed by IT instillation of LPS (Injury+LPS) or injured animals administered IT saline (Injury+vehicle). Second hit animals have increased IL-6 (A, n=6), CXCL1 (B, n=5) and MIP-2 (C, n=5) levels when compared to animals given IT LPS or injured animals administered IT saline. Data shown is the mean ± SEM. Differences are significant (p≤0.05) between all groups; the exception is MIP-2 levels in Injury+vehicle compared to LPS alone where no significant difference (p>0.05) is observed.

Figure 4. HTS resuscitation reduces the neutrophil response to a second hit

We compared the neutrophil response of injured animals resuscitated with HTS or LR prior to a second hit with IT LPS. Second hit animals resuscitated with HTS show a significant decrease (*p≤0.001) in BAL neutrophilia (A) and lung MPO activity (B) when compared to second hit animals without resuscitation (Injury+LPS). There is no significant effect of LR resuscitation (p>0.05) on BAL neutrophilia or MPO activity. Data shown is the mean ± SEM (n=5).

Figure 5. HTS resuscitation reduces the systemic innate immune response to a second hit

We compared the serum levels of IL-6, CXCL1, MIP-2 and CD11b expression in injured animals resuscitated with HTS or LR prior to a second hit with IT LPS. HTS resuscitated animals have a significant decrease (*p≤0.05) in serum IL-6 (A, n=8), MIP-2 (C, n=5) and CD11b expression (D, n=5) compared to second hit animals. There is no significant change (p>0.05) in CXCL1 by either resuscitation strategy (B, n=5, 8, 8 for Injury+LPS, HTS, and LR, respectively). LR resuscitated animals are also not significantly changed (p>0.05). Data shown is the mean ± SEM.