Our results demonstrate that after a single exposure to clinically relevant concentrations of inhaled LPS, C57BL/6CD44−/− mice have significantly less macrophage recruitment to the lung. This defect in macrophage recruitment was associated with impaired in vitro production of TNF-α, altered migration in response to multiple chemoattractants, and reduced adhesion to endothelium. Taken together, these results show that recruitment of macrophages to the lung after exposure to inhaled LPS is, in part, dependent on CD44.
Primary defects in CD44-deficient macrophage migration to the lungs have been previously described following intranasal infection with Mycobacterium tuberculosis
). In that study, CD44-deficient mice had a 50% reduction in pulmonary macrophages 2 wk after infection, suggesting that CD44 mediates resistance to mycobacterial infection through recruitment of macrophages at the site of infection and phagocytosis of the organism (15
). Similarly, we demonstrate that 48 h after LPS inhalation the absolute number of total leukocytes remained unchanged between the groups, yet there was an overall defect in macrophage recruitment in C57BL/6CD44−/−
mice. While our study does not address live infection with E. coli
, our observations suggest that a defect in macrophage recruitment in response to E. coli
LPS could have a substantial impact on inflammatory lung injury associated with live pathogens. It is likely that a combination of surface receptors regulate both the recognition and clearance of live pathogens.
Our study demonstrates a CD44-dependent defect in both in vitro
production of TNF-α and macrophage recruitment to the lung. Our in vitro
observation that the macrophage cytokine response to LPS is minimally attenuated in CD44-deficient animals suggests that CD44 can directly modify tlr4-dependent responses. We speculate that a complex interaction between CD44 signaling and tlr4 signaling is required for the complete biologic response to inhaled LPS, as has been previously suggested with both bleomycin lung injury (16
) and tumor cell signaling (17
). Similarly, previous studies have shown that recognition of M. tuberculosis
is dependent on CD44 (15
), nucleotide-binding oligomerization domain 2 (NOD2), and TLRs (18
). It remains plausible that the initial LPS response is dependent on tlr4, while secondary signaling can be modified by CD44. In this study, we show elevated HA in lung lavage fluid in LPS-exposed CD44-deficient animals, supporting the role of CD44 in clearance of soluble HA. Increased level of HA in the lung has previously been associated with recruitment of macrophages into the lung after bleomycin-induced lung injury (19
). Furthermore, there is evidence in the literature suggesting that hyaluronan can act as an endogenous ligand of tlr4
). We speculate that elevated levels of HA could modify local tlr4-dependent signaling in response to LPS. In addition, previous studies demonstrate that high-molecular-weight hyaluronan can attenuate inflammatory response (4
). It remains unclear whether the fragment size of HA can regulate the biologic consequences of HA ligation to TLRs. However, in this study, we demonstrate that CD44-dependent recruitment of macrophages to the lung after inhaled LPS is primarily related to defects in macrophage migration and endothelial adhesion.
Reduced absolute numbers of macrophages in the lung were associated with alterations in the kinetics of neutrophil trafficking after inhaled LPS. In this study, CD44-deficient animals had reduced concentration of PMNs at 24 h and increased numbers of PMNs at 48 h. Reduced concentration of neutrophils in CD44-deficient mice 24 h after exposure could be explained by several mechanisms, including a reduction in regulatory macrophages recruited into the lung, impaired PMN migration (6
), reduced endothelial adhesion (23
), or reduced concentration of local chemokines. In this study, we did not observe biologically meaningful differences in either chemokines in lavage fluid or neutrophil migration in the absence of hyaluronan matrix in vitro
. However, alveolar macrophages, which are known to play a critical role in neutrophil recruitment and clearance after exposure to inhaled LPS (24
), were reduced. We speculate that an absolute reduction in alveolar macrophages contributes to the reduced numbers of neutrophils at this time point. In addition, CD44 is known to play a role in the clearance of apoptotic neutrophils (5
). However, in this study, we did not detect any differences in percentage of apoptotic neutrophils by flow cytometry and annexin V staining at 24 h (data not shown). In context of previous studies, our observations support that both the intensity and mechanism of lung injury dictate the modifying role of CD44. Recent work by Liang and coworkers supports the idea that when mice are exposed to very high doses of LPS associated with substantial lung injury, CD44 can act as a negative regulator of acute pulmonary inflammation (26
). These observations support divergent roles of CD44 depending on the severity of lung injury.
The role of CD44 in lung inflammation appears dependent on the exposure as well as the duration, intensity, and timing after environmental challenge. While our results demonstrate that macrophage recruitment to the lung after exposure to low levels of aerosolized LPS is dependent on intact CD44, while the production of proinflammatory cytokines by macrophages is only partially dependent on CD44. Importantly, our data support a primary defect in macrophage–endothelial adhesion as a mechanism to explain an absolute reduction in the numbers of macrophages recruited into the lung. Defects in endothelial adhesion were associated with reduced recruitment of macrophages after single exposure to both low levels of inhaled endotoxin and the chemokine, MCP-1. Thus, the biologic response to commonly encountered environmental levels of inhaled LPS is, in part, dependent on functional CD44.