Overexpression of sICAM-1 in the alveolar epithelial space results in an exaggerated inflammatory response and early death in Gram negative pneumonia

Abstract

Background: A sizeable body of data demonstrates that membrane ICAM-1 (mICAM-1) plays a significant role in host defense in a site-specific fashion. On the pulmonary vascular endothelium, mICAM-1 is necessary for normal leukocyte recruitment during acute inflammation. On alveolar epithelial cells (AECs), we have shown previously that the presence of normal mICAM-1 is essential for optimal alveolar macrophage (AM) function. We have also shown that ICAM-1 is present in the alveolar space as a soluble protein that is likely produced through cleavage of mICAM-1. Soluble intercellular adhesion molecule-1 (sICAM-1) is abundantly present in the alveolar lining fluid of the normal lung and could be generated by proteolytic cleavage of mICAM-1, which is highly expressed on type I AECs. Although a growing body of data suggesting that intravascular sICAM-1 has functional effects, little is known about sICAM-1 in the alveolus. We hypothesized that sICAM-1 in the alveolar space modulates the innate immune response and alters the response to pulmonary infection.

Methods: Using the surfactant protein C (SPC) promoter, we developed a transgenic mouse (SPC-sICAM-1) that constitutively overexpresses sICAM-1 in the distal lung, and compared the responses of wild-type and SPC-sICAM-1 mice following intranasal inoculation with K. pneumoniae.

Results: SPC-sICAM-1 mice demonstrated increased mortality and increased systemic dissemination of organisms compared with wild-type mice. We also found that inflammatory responses were significantly increased in SPC-sICAM-1 mice compared with wild-type mice but there were no difference in lung CFU between groups.

Conclusions: We conclude that alveolar sICAM-1 modulates pulmonary inflammation. Manipulating ICAM-1 interactions therapeutically may modulate the host response to Gram negative pulmonary infections.

Figure 1

Design of SPC-sICAM-1 transgene construct and transfection into MLE12 cell line. The transgene, sICAM-1, was placed under the control of the human SPC promoter (-3700 to +24 bp). The SV40 cassette provided intronic and polyadenylation sequences. The approximate locations of primers for genotyping transgenic mice are indicated (arrowheads) together with the PCR product (a). Function of the transgene was demonstrated by transient transfection of the transgene into MLE12 cells (b). sICAM-1 in the cell culture supernatants was measured at 24 and 48 hrs as shown (n = 3, * P < 0.05).

Figure 2

Characterization of SPC-sICAM-1 transgenic mice. Transgene-specific primers demonstrated lung-specific expression of the SPC-sICAM-1 transgene in the lungs, with no expression detected in wild-type mice (a). Protein expression in SPC-sICAM-1 in BALF was increased (~2 log fold) over wild type mice (b, *** P < 0.05 by t-test), but did not significantly affect total BALF protein or serum sICAM-1 expression (c, d). Western analysis of BALF demonstrates a larger protein (~100 kDA, black arrowhead) in SPC-sICAM-1 BALF not present in wild type BALF. The lighter bands (white arrowhead) represent endogenous processing of sICAM-1. Whole lung mince from a wild type mouse is included for comparison. Two representative mice are shown for SPC-sICAM-1 (F1 generation) and wild type mice (e).

Figure 3

Overexpression of sICAM-1 in the distal lung results in decreased survival after K. pneumoniae infection. SPC-sICAM-1 mice and wild-type controls were inoculated intranasally with 2500 CFU of K. pneumoniae on day 0 and the percentage of mice surviving was determined over time. At 10 days, survival was significantly decreased in the SPC-sICAM-1 mice compared with infected wild-type controls. * P = 0.0012 compared with wild-type control mice.

Figure 4

Increased systemic dissemination, but similar lung burden, occurs 24 hours after K. pneumoniae infection. SPC-sICAM-1 mice and wild-type mice were inoculated intranasally with 2500 CFU of K. pneumoniae. After 30 minutes (a) and 24 hours (b), the animals were euthanized, and K. pneumoniae CFU were determined in lung homogenates. The percentage of positive spleen cultures and CFU were determined at 24 hours(c, d). Data are expressed as CFU per milliliter (mean ± SEM; n = 3 at 30 minutes; n = 14 for wild type and n = 15 for SPC-sICAM-1 at 24 hours; * P < 0.05 compared with wild-type).

Figure 5

Increased pulmonary inflammation is observed at 24 hours after K. pneumoniae infection in SPC-sICAM-1 mice compared to wild-type mice. SPC-sICAM-1 mice and wild-type mice were intranasally inoculated with 250 CFU of K. pneumoniae. After 24 hours, the animals were euthanized, and whole lung lavage was performed. Whole lung lavage was also collected from mice inoculated with PBS, but not exposed to K. pneumoniae, for comparison. Total number of cells was determined by counting with a hemacytometer (A). Representative histologic sections of lungs in wild type (B) and SPC-sICAM-1 mice (C) 24 hours after infection. Data are expressed as mean ± SEM. (n = 6 in all groups; * P < 0.05 compared with wild-type).

Figure 6

Increased pulmonary inflammation in SPC-sICAM-1 mice after K. pneumoniae infection is due to recruitment of both mononuclear cells and neutrophils. SPC-sICAM-1 mice and wild-type mice were intranasally inoculated with 250 CFU of K. pneumoniae. After 24 hours, the animals were euthanized, and whole lung lavage was performed. Cells were examined by flow cytometry with a gating strategy to identify leukocyte subpopulations as described in materials and methods (a, representative plot, SPC-ICAM-1 at 24 hours). SPC-sICAM-1 had greater accumulation of neutrophils (b), AM (c), and monocytes (d), compared to wild-type at 24 hours, although the differences in AM and monocyte numbers did not reach statistical significance. Data are expressed as mean ± SEM. (n = 6 in all groups; * P < 0.05 compared to wild-type).

Figure 7

In vivo phagocytosis of labeled microbeads by AM is similar in SPC-sICAM-1 and wild-type mice. Mice were lightly anesthetized and intranasally inoculated with 5 × 10⁷ FITC-conjugated polystyrene microbeads (1.7 micron). After 1 hour, mice were euthanized, and AM were recovered by whole lung lavage. Cells were recovered by centrifugation and examined by flow cytometry. Data are expressed as mean ± SEM for the percentage of AM that have engulfed beads (A) and the percentage of cells ingesting 1, 2, or > 2 beads (B). (n = 5 for all groups; no significant differences between groups)

Figure 8

In vitro stimulation of AM with LPS and recombinant sICAM-1 results in a synergistic increase in TNFα and MIP-2 production from AM. AM isolated by whole lung lavage from normal wild-type mice were stimulated with LPS (100 μg/ml) and/or recombinant sICAM-1 (50 μg/ml). Both TNFα (A) and MIP-2 (B) were measured by ELISA of cell culture supernatant after a 24 hour incubation. Data are expressed as mean ± SEM. (n = 6 for all groups; * P < 0.05 compared to all other conditions).

Figure 9

K. pneumoniae infection of SPC-sICAM-1 mice may be associated with greater alveolar leak compared to wild type mice. SPC-sICAM-1 mice and wild-type mice were intranasally inoculated with 250 CFU of K. pneumoniae. After 6 and 24 hours, the animals were euthanized, and whole lung lavage was performed. Albumin was measured by ELISA of cell free supernatant. Data are expressed as mean ± SEM. (n = 6 for all groups).