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Am J Physiol Lung Cell Mol Physiol 290: L962-L970, 2006. First published December 22, 2005; doi:10.1152/ajplung.00352.2005
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Shedding of soluble ICAM-1 into the alveolar space in murine models of acute lung injury

Michael P. Mendez,1,2 Susan B. Morris,1 Steven Wilcoxen,1 Elizabeth Greeson,1 Bethany Moore,2 and Robert Paine, III1,2

1Pulmonary Section, Department of Veterans Affairs Medical Center, and 2Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, Michigan

Submitted 12 August 2005 ; accepted in final form 19 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Intercellular adhesion molecule-1 (ICAM-1; CD54) is an adhesion molecule constitutively expressed in abundance on the cell surface of type I alveolar epithelial cells (AEC) in the normal lung and is a critical participant in pulmonary innate immunity. At many sites, ICAM-1 is shed from the cell surface as a soluble molecule (sICAM-1). Limited information is available regarding the presence, source, or significance of sICAM-1 in the alveolar lining fluid of normal or injured lungs. We found sICAM-1 in the bronchoalveolar lavage (BAL) fluid of normal mice (386 ± 50 ng/ml). Additionally, sICAM-1 was spontaneously released by murine AEC in primary culture as type II cells spread and assumed characteristics of type I cells. Shedding of sICAM-1 increased significantly at later points in culture (5–7 days) compared with earlier time points (3–5 days). In contrast, treatment of AEC with inflammatory cytokines had limited effect on sICAM-1 shedding. BAL sICAM-1 was evaluated in in vivo models of acute lung injury. In hyperoxic lung injury, a reversible process with a major component of leak across the alveolar wall, BAL fluid sICAM-1 only increased in parallel with increased alveolar protein. However, in lung injury due to FITC, there were increased levels of sICAM-1 in BAL that were independent of changes in BAL total protein concentration. We speculate that after lung injury, changes in sICAM-1 in BAL fluid are associated with progressive injury and may be a reflection of type I cell differentiation during reepithelialization of the injured lung.

alveolar epithelial cell; differentiation; aquaporin-5; cell culture; CD54

INTERCELLULAR ADHESION MOLECULE-1 (ICAM-1) is an ~100-kDa protein belonging to the immunoglobulin superfamily that is involved in leukocyte trafficking and lymphocyte activation (37). ICAM-1 is a transmembrane protein that is the counterreceptor for the beta2-integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), found on leukocytes. ICAM-1 expression can be induced on most structural cells, including endothelial cells, fibroblasts, and epithelial cells, by inflammatory cytokines (37). In contrast, in the normal lung, ICAM-1 is constitutively expressed in abundance on the apical surface of type I alveolar epithelial cells (AEC), but not on type II AEC in the absence of inflammatory stimulation (9, 19, 32). Similar to markers such as aquaporin-5 (AQP5) and RTI40 (6, 27, 41), constitutive ICAM-1 expression is a feature of type I, but not type II, AEC in the normal lung. Furthermore, when type II cells are isolated from rats, ICAM-1 expression is induced under culture conditions favoring expression of type I AEC characteristics (9). Previously, we have shown that absence or neutralization of ICAM-1 on AEC in vivo impairs function, but not recruitment, of leukocytes in the setting of pneumonia due to Klebsiella pneumoniae (19). Moreover, blockade of ICAM-1 in vitro results in decreased macrophage mobility (20), phagocytosis, and killing of K. pneumoniae (19).

In addition to its expression on the cell surface of endothelial cells, ICAM-1 is shed from that surface by proteolytic cleavage and is found in serum as soluble ICAM-1 (sICAM-1) (24, 37). sICAM-1 has been reported in the bronchoalveolar lavage (BAL) of humans and rats (10, 20), but little is known regarding the source or significance of sICAM-1 in the alveolar lining fluid of normal or injured lungs. The present study was undertaken to confirm the presence of sICAM-1 in alveolar lining fluid, to establish that AEC are the likely source of sICAM-1, and to determine the effects of inflammation or injury on the levels of sICAM-1 in the alveolar lining fluid.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Specific pathogen-free 6- to 12-wk-old wild-type C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in isolator cages within the Animal Care Facilities at the Veterans Affairs Research Laboratories. Mice received food and water ad libitum. The animal care committees at the Veterans Affairs Medical Center (Ann Arbor, MI) and the University of Michigan approved the experimental protocols.

Processing of BAL. At appropriate times, BAL was performed in mice from experimental and control groups using previously described methods (1). BAL was performed using five 1-ml aliquots of PBS that were pooled. Typical return was 90–95% of instilled volume. BAL fluid was centrifuged at 500 g for 10 min at 4°C to remove whole cells. Subsequently, aliquots of BAL fluid were subjected to high-speed centrifugation (100,000 g) with an ultracentrifuge (Beckman) to remove membrane fragments. Supernatants were stored at –70°C for subsequent analysis of sICAM-1 by ELISA (Pierce Biotechnology, Rockford, IL) or Western blot. Protein and albumin levels in BAL fluid were determined using a Coomassie protein assay (Pierce Biotechnology) and albumin ELISA (Bethyl Laboratories, Montgomery, TX), respectively.

Isolation and culture of murine AEC. Murine type II AEC were isolated by the method originally described by Corti et al. (11), as described previously (30). Briefly, after the pulmonary vasculature was perfused free of blood with PBS, type II AEC were freed from the lung by enzymatic digestion with dispase (Worthington Biochemical, Lakewood, NJ) infused via the trachea. The collected cells were filtered successively through 100-, 40-, and 25-µm nylon mesh filters to create a single cell suspension. Contaminating leukocytes were bound with biotinylated anti-CD32 (Fc{gamma}R; 0.65 µg/million cells) and anti-CD45 (common leukocyte antigen, 1.5 µg/million cells; BD Pharmingen) and removed using a magnetic cell separator after addition of streptavidin-coated magnetic particles. Cells not bound with magnetic particles were recovered and suspended in culture media. Viability was >97% by trypan blue exclusion. The cells were plated overnight in 100-mm culture plates. The nonadherent cells, including type II AEC, were recovered and counted. Viability was >97% by trypan blue exclusion. AEC were then cultured in fibronectin-coated tissue culture plates (Corning) in DMEM (GIBCO, Grand Island, NY) supplemented with 10% FCS (GIBCO). To test for purity, after 1–2 days in culture, adherent cells were stained for vimentin and cytokeratin 8 and found to be >95% vimentin negative and >95% cytokeratin 8 positive.

For cell culture experiments, cells were plated in 24-well plates at 0.5 x 106 cells/well or 12-well plates at 1.0 x 106 cells/well in DMEM supplemented with 10% FCS, penicillin, and streptomycin (GIBCO). Cells were allowed to adhere and became confluent over 72 h. For cytokine stimulation experiments, cells were stimulated with medium alone or with media supplemented with TNF-{alpha} or IL-1beta (0.1, 1, 10, or 100 ng/ml in each instance, both R&D Systems, Minneapolis, MN). After 48 h, supernatants were collected and exposed to high-speed (100,000 g) centrifugation to remove membrane fragments before measurement of sICAM-1 by ELISA.

Western blot analysis. After 3–4 days in culture, AEC monolayers in 35-mm dishes were washed with PBS. Cells were lysed using RIPA buffer. After 500 g centrifugation, the protein content of the supernatant was determined using a Coomassie protein assay (Pierce Biotechnology). The samples were denatured in sample buffer (2% SDS, 10% glycerol, and 62.5 mM Tris·HCl, pH 6.8) at 100°C and separated by SDS-PAGE (7.5% or 15% acrylamide) under nonreducing conditions, with loading of 20 µg of protein in each lane. After PAGE, the separated proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Richmond, CA). Full-range protein molecular weight standards were purchased from Bio-Rad. The PVDF membranes were incubated in 5% bovine serum albumin to block nonspecific binding and exposed to rat MAb AB796 (specific for the extracellular domain of mouse ICAM-1; R & D Systems), control rat IgG2B antibody (R&D Systems), goat antibody to AQP5 (specific for COOH terminus of mouse AQP5, Santa Cruz Biotechnology), or control goat IgG (Santa Cruz Biotechnology). The membranes were then incubated with anti-rat secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) or anti-goat secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The membranes were washed extensively in Tris-buffered saline after each step. Subsequently, the blots were developed using a chemiluminescence system (ECL Western Blotting Detection System; Amersham, Arlington Heights, IL) according to the manufacturer's recommendations. Recombinant mouse sICAM-1 (StemCell Technologies) was used to confirm recognition of the antibody to sICAM-1 in our samples.

Sublethal hyperoxia. C57BL/6 mice were exposed to 95% oxygen for 1–5 days to induce lung injury. Mice experienced little or no mortality if returned to room air after 4 days. If allowed to recover in room air, they were indistinguishable from their room air-matched controls. Mice were exposed to hyperoxia in a Plexiglas chamber with a continuous oxygen monitor (Proox model 110; Biospherix, Lacona, NY). They continued to receive food and water ad libitum and were checked daily for evidence of disease. CO2 in the chamber was monitored and remained <5%. At the appropriate time points, mice were anesthetized with intraperitoneal injection of Euthazol (Diamond Animal Health) and exsanguinated by sectioning of the abdominal aorta. BAL was performed as described above.

Lung injury induced by FITC. It has been shown previously that intratracheal inoculation of mice with FITC results in acute lung injury, which subsequently progresses to pulmonary fibrosis (8). For these studies, mice were anesthetized with pentobarbital sodium (Abbott Laboratories, North Chicago, IL) at 50 mg/kg ip. The trachea was exposed, and 50 µl of FITC solution were instilled using a 26-gauge needle. FITC solution was prepared by adding 84 mg of FITC (F-7250; Sigma, St. Louis, MO) to 10 ml of sterile PBS, which was then vortexed and sonicated. Control mice were inoculated with the same volume of sterile PBS. The skin incision was closed with 4-0 surgical prolene stitches. Mice were allowed to recover from the procedure and were returned to appropriate housing. At the appropriate time points, mice were anesthetized with intraperitoneal injection of Euthazol and exsanguinated by sectioning of the abdominal aorta. BAL was performed as above.

Immunohistochemistry for ICAM-1. Lungs were fixed and inflated with equivalent volumes of buffered formalin. Paraffin-embedded mouse lung tissue was cut on a microtome, and 4-µm-thick sections were placed on glass microscope slides for immunohistochemistry. Sections were deparaffinized and hydrated by passing through a series of xylene and graded alcohols. Immunohistochemistry was performed using the goat ABC Staining System (Santa Cruz Biotechnology). Endogenous peroxidase activity was quenched by exposure to 3% H2O2 in PBS for 10 min. Nonspecific binding of antibodies to the tissues was blocked by incubating the tissue with 1.5% donkey serum (Santa Cruz Biotechnology) in PBS for 30 min. For the detection of ICAM-1, goat anti-mouse ICAM-1 antibody was used (AF796; R&D Systems) at a titer of 1:50. Control goat IgG (Santa Cruz Biotechnology) at a similar concentration was used. A biotinylated donkey anti-goat IgG (Santa Cruz Biotechnology) was used at a titer of 1:200. Tissues were incubated with primary antibodies overnight at 4°C. After being washed, tissues were incubated with secondary antibody for 30 min. 3,3'-Diaminobenzidine (Santa Cruz Biotechnology) was used as peroxidase substrate. In each instance, sections from different time points were processed together, with equal time for color development. Tissues were counterstained with hematoxylin (Santa Cruz Biotechnology). Stained tissues were mounted in Permount (Fisher Chemical) and photographed using a Nikon Labphot 2 microscope with Ektachrome film (Kodak).

Statistical analysis. Data are expressed as means ± SE and were compared, using a two-tailed Student's t-test or ANOVA if more than two groups were compared, with the InStat software package from GraphPad Software (San Diego, CA). Differences were considered statistically significant if P values were <0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
sICAM-1 is present in normal alveolar lining fluid. To determine the presence of sICAM-1 in the alveolar lining fluid of normal mice, whole lung lavage was performed on C57BL/6 mice. sICAM-1 was detected in lavage fluid in all mice. Despite dilution of alveolar lining fluid by the lavage procedure, concentrations of sICAM-1 in lung lavage were 386 ± 50 ng/ml after high-speed centrifugation. Levels of sICAM-1 measured with ELISA were unchanged by centrifugation at either low speed (500 g) or high speed (100,000 g) to remove ICAM-1 associated with whole cells or with membrane fragments, respectively. These data indicate that only the soluble form, not membrane-associated ICAM-1, was present in the alveolar lining fluid (Fig. 1A). Western blot analysis confirmed the presence and size of sICAM-1 in BAL fluid recovered from normal mice. For comparison, cell lysates from type II AEC isolated from normal mice and placed in culture for 3 days (to allow induction of membrane-associated ICAM-1) were also evaluated. Typically, sICAM-1 is generated by proteolytic cleavage of the membrane-bound protein from the cell surface (40). As might be anticipated, the size of BAL sICAM-1 (~90 kDa) was lower than the bands representing membrane-associated ICAM-1 (~105–110 kDa) (Fig. 1B).


Figure 1
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  		Fig. 1. Measurement of soluble ICAM-1 (sICAM-1) in bronchoalveolar lavage (BAL) of normal mice. BAL was collected as described in MATERIALS AND METHODS. Samples were exposed to no-spin, 500 g centrifugation for 10 min and 100,000 g centrifugation for 1 h. sICAM-1 levels were measured by ELISA (A). Data are expressed as means ± SE. n = 3 in a representative experiment. The size of sICAM-1 was confirmed by Western blot analysis of BAL in normal mice (B). We confirmed that this antibody recognized recombinant sICAM-1 (not shown).

 
sICAM-1 shedding by AEC in culture. Previously, we have shown that ICAM-1 expression is induced as isolated rat type II AEC spread in culture and assume characteristics of the type I cell phenotype (9). Like their rat counterparts, murine type II cells that are isolated and placed in cell culture express little ICAM-1 initially. However, as the AEC spread in culture, ICAM-1 in cell lysates increased, remaining relatively constant from days 3 to 7 in culture (Fig. 2A). Similarly, expression of the type I AEC marker AQP5 increased after day 1 and reached maximal expression at day 7 (Fig. 2B). To determine whether AEC shed sICAM-1 spontaneously in vitro, we measured sICAM-1 in culture supernatants collected over 48 h beginning on day 3 or day 5 after isolation. sICAM-1 was present in the culture medium collected at the earlier time point and increased significantly (387.4 ± 82 ng/ml vs. 651.2 ± 14 ng/ml) as AEC continued to spread in culture (Fig. 2C). These data confirm AEC as a source of sICAM-1 and indicate that AEC transition toward the type I cell phenotype is associated with increased sICAM-1 release.


Figure 2
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  		Fig. 2. Differentiation of cultured murine alveolar epithelial cells (AEC) and measurement of sICAM-1 shedding in the culture supernatant. AEC were isolated as described in MATERIALS AND METHODS, and monolayers of AEC were established. Expression of membrane-associated ICAM-1 on isolated AEC in culture was determined throughout days 1–7. Cells were lysed in RIPA buffer and analyzed by Western blot. Increased cell-associated ICAM-1 was seen after day 1 in culture (A). Similarly, cell lysates demonstrated increased expression of aquaporin-5 (AQP5) after cells had spread in culture (B). In separate experiments, after cells were allowed to adhere for 48 h, cells were gently washed with PBS, and fresh media supplemented with serum were added. To examine the time course of shedding, media were collected after 48 h (days 3–5) or replaced with fresh media and collected after 48 more hours (days 5–7). Supernatants were collected and subjected to high-speed centrifugation before analysis by ELISA (C). Data are expressed as means ± SE. *P < 0.03 vs. days 3–5; n = 3.

 
Effect of inflammatory cytokines on sICAM-1 shedding by AEC in culture. Endothelial cells are induced to express ICAM-1 in the setting of inflammation and release sICAM-1 in response to inflammatory cytokines (38). Previously, we have shown that membrane-associated ICAM-1 expression on rat AEC is minimally affected by stimulation with inflammatory cytokines in vitro (2). To determine whether inflammatory cytokine stimulation influenced sICAM-1 shedding from murine AEC in culture, we exposed AEC to a 1,000-fold range of serial dilutions of two cytokines that are found in the inflamed lung, TNF-{alpha} and IL-1beta, and measured sICAM-1 in the culture supernatants. There were no significant differences in the levels of sICAM-1 after TNF-{alpha} stimulation over the entire range of doses (Fig. 3). Modest increases (1.2- to 1.5-fold) in sICAM-1 shedding were observed with IL-1beta stimulation at doses of 1 ng/ml and above. Thus sICAM-1 shedding by AEC in vitro is moderately altered by IL-1beta but not by other signals associated with acute inflammation.


Figure 3
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  		Fig. 3. Effects of inflammatory mediators on AEC shedding of sICAM-1 in vitro. Primary cultures of AEC were established as described in MATERIALS AND METHODS. Cells were allowed to adhere for 48 h, followed by gentle washing with PBS and replacement of media with or without cytokines. Supernatant was collected and subjected to high-speed centrifugation before measurement by ELISA. There were increased levels of sICAM-1 in the culture supernatant as a result of IL-1beta stimulation at higher doses after 48 h as opposed to TNF-{alpha} stimulation, which resulted in no difference at any dose studied. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. control; **P < 0.01 vs. control.

 
sICAM-1 shedding in murine models of lung injury. Having determined that inflammation alone had limited effect on sICAM-1 shedding in vitro, experiments were performed to determine the impact of acute lung injury on sICAM-1 levels in the alveolar lining fluid. Two models of injury were examined. In the first model, mice were exposed to hyperoxia (FIO2 >95%) for 5 days. Mice exposed to hyperoxia for >4 days exhibit a pattern of diffuse lung injury that ultimately leads to death if the mice are not returned to normoxia (1). However, if mice are returned to normoxia after 4 days, there is complete recovery and no mortality (3). sICAM-1 levels in BAL fluid were determined after 1, 2, 3, or 5 days of hyperoxia. Age-matched mice in room air served as controls. sICAM-1 levels increased significantly after 5 days in hyperoxia compared with control (984.6 ± 308 ng/ml vs. 353.2 ± 68 ng/ml) (Fig. 4A). Concomitantly, protein (Fig. 4B) and albumin concentration of the BAL also increased, confirming loss of barrier function of the alveolar wall. Interestingly, when sICAM-1 was normalized to albumin in BAL fluid to control for the leak of serum proteins into the alveolar space (Fig. 4C), this value fell below that of control mice at days 3 and 5 of hyperoxia, suggesting a vascular source of increased sICAM-1.


Figure 4
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  		Fig. 4. Shedding of sICAM-1 in the alveolar lining fluid during hyperoxic lung injury. Mice were exposed to hyperoxia for 1, 2, 3, and 5 days; whole lung lavage was then performed, and sICAM-1 protein was measured in the BAL fluid (after high-speed centrifugation) by ELISA (A). Data are expressed as means ± SE. *P < 0.001 vs. control; **P < 0.05 vs. control. Levels of sICAM-1 and total protein were significantly increased after 3 days of hyperoxia (B). *P < 0.001 vs. control; n = 5 in each group. Serum levels of sICAM-1 were not significantly different from normal controls at any time point (data not shown). The ratio of sICAM-1 to albumin was determined at each time point (C).

 
Having evaluated a model of acute lung injury associated with complete recovery, we next evaluated sICAM-1 in BAL fluid in a model of lung injury that progresses to pulmonary fibrosis (8). C57BL/6 mice were intratracheally inoculated with FITC or saline control. sICAM-1 levels in BAL fluid increased over control levels throughout days 1–4 (Fig. 5A). In contrast, the initial protein leak caused by lung injury peaked on day 1, when inflammation was maximal, and continued to resolve on subsequent days (Fig. 5B). Thus sICAM-1 normalized to albumin in BAL fluid increased markedly on days 3 and 4 following injury. This delayed increase in BAL sICAM-1 suggests that an intra-alveolar source of sICAM-1, and not serum leak, contributes a major component of sICAM-1 to the alveolar lining fluid in the setting of lung injury (Fig. 5C). Western blot analysis for ICAM-1 in BAL fluid 4 days post-FITC confirms that the size of sICAM-1 is consistent with that found in normal BAL fluid. Thus these data suggest that progressive lung injury can directly lead to significant increases in local release of sICAM-1 into the alveolar lining fluid.


Figure 5
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  		Fig. 5. Shedding of sICAM-1 in the alveolar lining fluid after lung injury due to FITC. Mice were intratracheally inoculated with FITC, and whole lung lavage was performed. sICAM-1 protein was measured in the BAL fluid (after high-speed centrifugation) by ELISA. Levels of sICAM-1 were significantly increased in the BAL after inoculation (A). Data are expressed as means ± SE. *P < 0.001 vs. day 0; **P < 0.001 vs. day 1. Total protein in BAL fluid increased after the initial injury with a subsequent decline (B). +P < 0.001 vs. day 0; ++P < 0.05 vs. day 4; n = 3 in each group. The ratio of sICAM-1 to albumin was determined at each time point (C). The size of sICAM-1 was confirmed by Western blot analysis of BAL in FITC-injured mice (postintratracheal day 4) and compared with normal BAL and membrane-associated ICAM-1 (D).

 
Immunohistochemical localization of ICAM-1in FITC-induced lung injury. As expected, immunohistochemical staining of ICAM-1 in normal mouse lungs demonstrates intense linear staining (brown) along alveolar walls (Fig. 6, A and B). After FITC instillation, there is evidence of thickened alveolar interstitium (day 2, Fig. 6C). Evidence of increasing injury was observed on days 3 and 4 (Fig. 6, D and E). Panels show representative areas of injury. ICAM-1 staining is noted throughout the period of injury in areas of injury and areas that are relatively spared (not shown).


Figure 6
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  		Fig. 6. Immunohistochemical localization of ICAM-1 expression in FITC-induced lung injury. Lungs were harvested from mice after intratracheal instillation of FITC: normal (A, B, and F), day 2 (C), day 3 (D), and day 4 (E, G). Formalin-fixed, paraffin-embedded lungs were sectioned and stained for immunohistochemistry with a polyclonal antibody specific for ICAM-1 (A–E) and counterstained. Panels show representative areas of injury. Dark brown staining represents ICAM-1. Control IgG antibody demonstrated no staining (F, G). Bronchial epithelium (be) and artery (ar) are labeled for orientation in B. Evidence of acute lung injury with thickened alveolar interstitium is demonstrated on day 2 (C, arrowhead) with progression of cellular infiltrate on days 3 (D) and 4 (E, G). Magnification, x20 (A), x40 (B–E, G), and x100 (F).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this article, we report the expression of sICAM-1 in the alveolar lining fluid of normal mice and after lung injury. Significant quantities of sICAM-1 are present in the alveolar lining fluid of normal mice, as measured in BAL fluid. Moreover, sICAM-1 is found in culture supernatants of murine AEC in primary culture as type II cells spread and assume characteristics of the type I cell phenotype, suggesting that type I AEC are likely to be a major source of BAL sICAM-1. Inflammatory mediators had at most a modest effect on shedding of sICAM-1 by AEC in vitro. In two different animal models, lung injury increased sICAM-1 levels in alveolar lining fluid. However, the temporal pattern of alveolar permeability and sICAM-1 shedding as measured by an index of sICAM-1/albumin (ng/µg) suggests differing sources of the increased sICAM-1 levels in the alveolar lining fluid. Increasing sICAM-1 in the BAL fluid in the setting of resolving leak suggests that a major source of sICAM-1 is the alveolar epithelium, beyond any contribution of leak from the vascular compartment. These studies will help construct a framework for the evaluation of sICAM-1 shedding in the alveolar lining fluid and provide preliminary evidence that the levels of sICAM-1 in the alveolar lining fluid may have significance as a marker for the health of the alveolar epithelium.

Membrane-associated ICAM-1 plays an important role as a costimulatory molecule in immune cell function and as an adhesion molecule in leukocyte migration to sites of inflammation (36, 37). In the lung, ICAM-1 has been shown to be important in the modulation of leukocyte behavior (28, 29), mobility (29), adhesion (4), and recruitment (4, 21). Previously, using electron microscopy, different groups have demonstrated that ICAM-1 is expressed on the apical surface of type I AEC in the normal lung, but not on type II AEC in the absence of inflammation (7, 19, 32). Our laboratory has shown that freshly isolated rat type II AEC in culture do not express ICAM-1 (9). As the cells adhere and spread in culture, they assume features of the type I cell phenotype, including abundant expression of ICAM-1 (9). We now report that murine AEC in primary culture undergo this same process. As the cells spread in culture, expression of ICAM-1 and AQP5, molecules found on the surface of type I AEC in vivo, is induced.

To confirm that AEC are a likely source of sICAM-1 in the alveolar lining fluid, we used an in vitro system that takes advantage of the fact that type II AEC are progenitors of type I AEC and that expression of specific type I cell characteristics is induced when type II cells are isolated and allowed to spread in culture (31). Although quantitative differences in level of expression of specific genes remain between these cultured AEC and freshly isolated type I cells (15), this model has proved to be an effective tool for studying the regulation and activity of individual AEC phenotypic characteristics. We found increased levels of sICAM-1 in the supernatant at later time points. At the same time, Western blot analysis of AEC lysates revealed that membrane-associated ICAM-1 expression increased by day 3 in culture but remained relatively constant over days 3–7. Thus the in vitro data demonstrate constitutive shedding of sICAM-1 from AEC that are undergoing transition toward the type I AEC phenotype and suggest that shedding of sICAM-1 may be a process that is a normal function of type I AEC. Because sICAM-1 release into the culture supernatant continued to increase even as cell-associated ICAM-1 remained relatively stable, it is likely that sICAM-1 shedding is a regulated process that is not simply a consequence of cell surface ICAM-1 expression.

Inflammatory cytokines such as TNF-{alpha}, IFN-{gamma}, or IL-1 can induce ICAM-1 expression and sICAM-1 shedding from cultured human umbilical vein endothelial cells, lung fibroblasts, aortic smooth muscle cells, dermal microvascular endothelial cells, bronchial epithelial cells, and keratinocytes (23, 37, 38). A putative signaling pathway mediating cleavage of membrane-associated ICAM-1 to form sICAM-1 in endothelial cells is the Ras-Raf-MAPK cascade (38). However, we are not aware of any data regarding signaling pathways in AEC. Our laboratory has previously shown that inflammatory mediators have little effect on ICAM-1 expression on AEC in response to these cytokines (2). We now show that IL-1beta has a modest effect on sICAM-1 shedding by AEC in vitro, whereas TNF-{alpha} does not influence sICAM-1 release over a wide range of doses. Inflammatory cytokines may not play a prominent role in sICAM-1 shedding, given our observation that time in culture has a more pronounced effect on the quantity of sICAM-1 released than high-dose IL-1beta.

sICAM-1 is found in normal serum, with considerable variation in glycosylation (36, 37). The source of sICAM-1 in serum is most likely proteolytic cleavage of membrane-associated ICAM-1 (12) on endothelial cells (38). Several clinical studies have described increased serum levels of sICAM-1 in a variety of inflammatory settings, including heart disease (22, 33), acute lung injury (14), and radiation pneumonitis (18), leading to the hypothesis that sICAM-1 levels may reflect disease activity.

Studies correlating serum sICAM-1 to disease have led to investigation of this molecule in the lung as a marker of disease. Increased sICAM-1 in BAL has been described in adults with granulomatous lung diseases such as sarcoidosis, tuberculosis, hypersensitivity pneumonitis, and radiation pneumonitis (3, 10, 18) and in children exposed to second-hand smoke (16). Kasper et al. (20) and Beck-Schimmer et al. (5) have shown that a soluble form of ICAM-1 can be measured in the BAL of rats by Western blot analysis after immunoprecipitation and by ELISA, respectively. We now demonstrate that sICAM-1 can be measured in the alveolar lining fluid of normal mice. By subjecting the BAL to high-speed centrifugation, we confirmed that the vast majority of ICAM-1 in the alveolar lining fluid of normal mice and in the setting of lung injury is true sICAM-1 rather than ICAM-1 associated with membrane fragments.

Researchers have sought a specific marker of lung injury in the alveolar lining fluid that may aid in the diagnosis or prognosis of the disease (10, 25, 26). Conner et al. (10) have reported that patients with acute respiratory distress syndrome have increased levels of sICAM-1 in lung edema fluid compared with serum. Levels are also higher compared with patients with cardiogenic pulmonary edema. However, there is no information regarding the source of sICAM-1 in the setting of acute lung injury, nor is there information to allow comparison with normal controls. For this reason, we chose to examine the pattern of sICAM-1 shedding in the alveolar lining fluid of mice in models of lung injury.

We examined two models of lung injury, hyperoxia and intratracheal instillation of FITC. Both of these insults cause a temporally and spatially distinct pattern of lung injury. Intratracheally inoculated FITC deposits into the distal air spaces and causes abrupt lung injury with transient leak that eventually leads to fibrosis in survivors (8). Histologically, the lungs demonstrate alveolar wall edema, alveolar exudates, and acute inflammation 1 day after intratracheal FITC inoculation (8). In contrast, exposure to hyperoxia causes a more slowly developing lung injury from which mice may recover if returned to normoxia (1). One day after FITC inoculation, we found an increase in protein concentration in the BAL fluid, suggesting that leak from acute lung injury had occurred. Protein and albumin levels decreased at later time points. Interestingly, levels of sICAM-1 in BAL fluid increased significantly throughout the observed time course (days 1, 2, and 5), even as leak resolved. This suggests that the source (vascular leak vs. alveolar shedding) of sICAM-1 accumulation in the alveolar lining fluid depends on the mechanism and temporal pattern of injury.

There are several potential sources contributing to increased sICAM-1 release into the alveolar lining fluid in the setting of lung injury. Individual type I AEC might be stimulated to release a greater fraction of cell surface ICAM-1 as sICAM-1. Immunoelectron micrographic data from Kang et al. (19) showing decreased cell-associated ICAM-1 per nanometer of cell surface of type I cells after hyperoxia might support this hypothesis. Second, assays were performed after 100,000-g centrifugation to remove membrane fragments; therefore, the increase in sICAM-1 in BAL fluid after lung injury is unlikely to be attributable to fragments from damaged type I cells in the alveolar lining fluid. Another possible explanation is that, in the injured lung, additional cells such as alveolar macrophages or type II cells express ICAM-1 (19) and thus may contribute sICAM-1 to the alveolar lining fluid. A final explanation, which is consistent with our in vitro data, involves type II cells that spread and differentiate to form new type I cells that replace cells lost to injury. We found that sICAM-1 in the culture supernatant increased as cells spread over time in culture. It is possible that increased sICAM-1 is released during this differentiation process. Additionally, the immunohistochemical data (Fig. 6) show intense ICAM-1 staining throughout the observed course of FITC-induced injury (days 1–4) that is localized to the alveoli, suggesting the alveolar cells as a likely source.

The enzymes responsible for release of sICAM-1 from the cell surface of type I AEC have not yet been identified. In other cell types, there is evidence that matrix metalloproteases mediate sICAM-1 shedding (13, 24). Because matrix metalloproteases are normally secreted by alveolar macrophages (35) and also associated with the membrane of macrophages (17), sICAM-1 levels may be modulated by the interaction of alveolar macrophages with AEC as they move throughout the alveolar wall. However, on the basis of our in vitro observations, basal shedding does not involve the influence of another cell type. However, after lung injury, activation of alveolar macrophages may contribute to increased shedding of sICAM-1 as they interact with AEC. Further studies will be required to determine which of these mechanisms contribute to the increase in sICAM-1 in alveolar lining fluid after lung injury.

There is limited information regarding the biological effects of sICAM-1 in the airway compartment. Under experimental conditions, sICAM-1 directly activates alveolar macrophages in vitro (35). These investigators also found that intratracheal instillation of sICAM-1 enhanced lung injury induced by IgG immune complexes generated in vivo (34). Conversely, AEC cell-associated ICAM-1 plays an important role in alveolar macrophage migration and phagocytic activity at the alveolar surface (28). We can speculate that sICAM-1 in the alveolar lining fluid may block binding of membrane-associated ICAM-1 to beta2-integrins and thus decrease inflammatory cell adhesion to the epithelial cell surface. By modulating this adhesive interaction, sICAM-1 may limit injury to epithelial cells or may facilitate release of macrophages from the epithelial cell surface, thus promoting alveolar macrophage mobility within the alveolar space. Finally, ICAM-1 is the receptor by which rhinovirus enters epithelial cells (39). Expression of ICAM-1 on the normal type I AEC would then put the host at risk of lung injury in the setting of the common cold. Expression of sICAM-1 within the alveolar lining fluid may decrease rhinovirus binding to type I cells and prevent internalization and infection of AEC (39).

In summary, our studies demonstrate that sICAM-1 is a normal constituent of the alveolar lining fluid and is shed by AEC. In culture, AEC shed increasing amounts of sICAM-1 as they assumed type I AEC features. It does not appear that inflammation alone can account for modulation of sICAM-1 shedding in AEC; however, after lung injury, there are increased levels of sICAM-1 in the alveolar lining fluid. These studies indicate that sICAM-1 may be useful as an indicator of the health of the alveolar epithelium.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants (to R. Paine III) from the Department of Veterans Affairs (VA Merit Review) and National Institutes of Health (NIH) Grant HL-64558 and by a research training fellowship Grant NIH T32 (to M. P. Mendez).


   ACKNOWLEDGMENTS
 
The authors thank Dr. Paul J. Christensen, Dr. Timothy Vollbrecht, and Emily Damuth for critically reviewing the manuscript and the members of the Ann Arbor Veterans Affairs Medical Center Research Enhancement Award Program for helpful suggestions and discussion.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Paine III, Pulmonary Section (111G), Veterans Affairs Medical Center, 2215 Fuller Road, Ann Arbor, MI 48105 (e-mail: rpaine{at}umich.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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