CD44 deficiency leads to enhanced neutrophil migration and lung injury in Escherichia coli pneumonia in mice

Abstract

CD44 is a major cell-surface receptor for hyaluronic acid (HA), a glycosaminoglycan component of extracellular matrix. HA-CD44 interactions have been implicated in leukocyte extravasation into an inflammatory site. This study examined the role of CD44 in acute inflammatory responses during pneumonias induced by Escherichia coli and Streptococcus pneumoniae using CD44-deficient mice. In E. coli-induced pneumonia, neutrophil accumulation in the lungs and edema formation was increased by 84% and 88%, respectively, in CD44-deficient mice compared to wild-type mice. In contrast, no difference was observed between these genotypes in S. pneumoniae-induced pneumonia, and the HA content in the lungs decreased after instillation of S. pneumoniae, but not E. coli, in both genotypes. Studies to determine the mechanisms for this enhanced response showed that: 1) neutrophil apoptosis was not different between these two genotypes in either type of pneumonia; 2) CD44 deficiency resulted in enhanced mRNA expression of several inflammatory genes; and 3) CD44-deficient neutrophils migrated through Matrigel in response to chemoattractants faster and in greater numbers than wild-type neutrophils in vitro and this increase was in part dependent on HA content in the Matrigel. These data demonstrate that CD44 deficiency results in enhanced inflammation in E. coli but not S. pneumoniae-induced pneumonia, suggesting a previously unrecognized role for CD44 in limiting the inflammatory response to E. coli.

Figure 1.

Histological sections of lungs of wild-type (a, c) and CD44-deficient mice (b, d) after 6 hours of pneumonia induced by E. coli (a, b) or S. pneumoniae (c, d). Original magnification, ×600.

Figure 2.

Neutrophil accumulation (A) and edema formation (B) in the lungs of CD44-deficient mice and wild-type mice after 6 hours of pneumonia induced by E. coli or S. pneumoniae. A: The neutrophil accumulation was quantified by point counting as described in Material and Methods. The data are presented as percent distal lung volume occupied by neutrophils. B: Edema formation was evaluated by measuring the accumulation of EVA as described in Material and Methods. Open bars, wild-type mice; filled bars, CD44-deficient mice. All of the data are presented as mean ± SEM (n ≥ 5 in each group). *, P < 0.05 when compared to wild-type mice.

Figure 3.

Bacteria recovered after 6 hours of pneumonia induced by E. coli or S. pneumoniae in the lungs of CD44-deficient mice and wild-type mice. The number of CFU of either bacterium instilled was determined. After 6 hours of pneumonia, the lungs were homogenized, and the number of CFU of either bacterium in the lung homogenate was measured. Open bars, wild-type mice; filled bars, CD44-deficient mice. The data are presented as number of CFU of bacteria instilled or recovered (n = 5 in each group).

Figure 4.

Percentage of apoptotic neutrophils recovered in the BAL fluid of wild-type or CD44-deficient mice 6 hours after instillation of E. coli or S. pneumoniae. Apoptotic neutrophils in the BAL fluid were evaluated by annexin V and Gr-1 staining and quantified by flow cytometry as described in Material and Methods. The data are expressed as the percentage of neutrophils that are annexin V-positive and presented as mean ± SEM (n = 5 or 6 in each group). Open bars, wild-type mice; filled bars, CD44-deficient mice.

Figure 5.

The distance that neutrophils migrated into Matrigel after 30 minutes. CD44-deficient and wild-type leukocytes isolated from venous blood were labeled with calcein AM and added to transwell filters coated with Matrigel. The number of neutrophils that migrated into the gels was counted from the top of the gels toward the chemoattractant at increments of 50 μm. For each experiment, the number of neutrophils in five fields (×200) was counted and averaged at each incremental level. Data are presented as mean ± SEM from five or six separate experiments. Open symbols, wild-type neutrophils; filled symbols, CD44-deficient neutrophils. The average distance that neutrophils migrated into Matrigel was calculated and included in the graph. *, P < 0.05 when compared with wild-type neutrophils.

Figure 6.

The number of neutrophils that migrated across Matrigel after 6 hours. CD44-deficient and wild-type leukocytes were isolated from venous blood and added to transwell filters coated with Matrigel. The percentage of neutrophils that migrated through the transwells in response to either buffer or fMLP placed in the bottom chambers was calculated. Open bars, buffer in the lower chamber; filled bars, 10⁻⁷ mol/L fMLP in the lower chamber. The data were presented as percent neutrophils migrated and was presented as means ± SEM (n ≥ 8 in each group). *, P < 0.05 when compared to buffer; #, P < 0.05 when compared to wild-type neutrophils.

Figure 7.

The effect of treatment of Matrigel with hyaluronidase on the migration of wild-type and CD44-deficient neutrophils. Matrigel at a higher concentration (178 μg/well) or a lower concentration (125 μg/well) was treated with 10 U/ml hyaluronidase or control vehicle as described in Material and Methods. The number of neutrophils that migrated across Matrigel in response to fMLP after 6 hours was measured as described above. Open bars, treatment of Matrigel with control vehicle; filled bars, treatment of Matrigel with hyaluronidase. The data were presented as percent neutrophils migrated and was presented as means ± SEM (n ≥ 5 in each group). *, P < 0.05 when compared to treatment with control vehicle; #, P < 0.05 when compared to wild-type neutrophils.

Figure 8.

HA content in the lungs of CD44-deficient mice and wild-type mice after instillation of saline, E. coli, or S. pneumoniae. HA content was measured as described in Materials and Methods. White bars, saline-treated mice; gray bars, E. coli-treated mice; black bars, S. pneumoniae-treated mice. The data are means ± SEM (n = 3). *, P < 0.05 when compared to their respective saline-treated control mice.

Figure 9.

Inflammatory gene expression in the pneumonic lungs of CD44-deficient mice and wild-type mice after 6 hours of pneumonia induced by E. coli or S. pneumoniae. Expression of mRNAs from whole lungs was detected in Northern blots as described in Materials and Methods. Aldolase was used as a loading control. Lanes 1 to 6: mRNA in lungs of wild-type and CD44-deficient mice after saline (lanes 1 and 2), S. pneumoniae (lanes 3 and 4), and E. coli (lanes 5 and 6), respectively. Data shown is representative of three independent experiments.