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Published ahead of print on May 1, 2003, doi:10.1165/rcmb.2002-0123OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 506-512, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0123OC

Neutrophil Adhesion Molecule Expression in the Developing Neonatal Rat Exposed to Hyperoxia

Susan M. Gerik, Susan E. Keeney, Dara V. Dallas, Kimberly H. Palkowetz and Frank C. Schmalstieg

Departments of Pediatrics and Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas

Address correspondence to: Susan E. Keeney, M.D., Department of Pediatrics, Route 0526, University of Texas Medical Branch, Galveston, TX 77555-0526. E-mail: skeeney{at}utmb.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neonatal rats have an increased tolerance to hyperoxia, which is associated with a diminished pulmonary inflammatory response compared with adults. To investigate this differing response, expression of the neutrophil adhesion molecules, L-selectin and CD18, and levels of soluble L-selectin, were examined using flow cytometry and sandwich enzyme-linked immunosorbent assay on air-exposed neonatal rat neutrophils at 0–24 and 72 h and 7, 10, 14, and 21 d of age compared with the adult and after exposure to hyperoxia (>= 98% O2) for 56 h in adults and for 72 h and 7 d in neonates. Expression of L-selectin in 0–24-h neonates was similar to adults, but was significantly lower than adults at 72 h and 7 d (P = 0.011). Soluble L-selectin levels were significantly higher than those in adults in the 0–24- and 72-h neonates (P < 0.001). CD18 expression in unstimulated and activated neutrophils of neonatal rats was higher at 0–24 h than in the adult (P < 0.001), but thereafter did not differ from adults. After hyperoxic exposure, L-selectin did not differ between the exposure groups but soluble L-selectin tended to increase in neonates after 7 d of O2 exposure Finally, CD18 was significantly higher after hyperoxic exposure of the adult (P = 0.008), but did not change with oxygen exposure in the neonate. Based on these findings, we speculate that differences between neonatal and adult rats in expression of L-selectin may contribute to delayed oxygen toxicity in neonatal rats.

Abbreviations: calf serum, CS • enzyme-linked immunosorbent assay, ELISA • formylmethionylleucylphenylalanine, FMLP • lipopolysaccharide, LPS • o-phenylenediamine, OPD • phosphate-buffered saline, PBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oxygen toxicity results in a pulmonary inflammatory process that has been well-described. Although high oxygen tension may be directly toxic to pulmonary tissue (1), pulmonary leukocyte accumulation amplifies the process of toxic oxygen metabolite production and lung damage (2). Neonates of several species have an increased tolerance to hyperoxia (3), but the mechanisms are incompletely understood. Not only may antioxidant-protective mechanisms play a role in this tolerance (3, 4), but the relative inability of the neonate to mount an inflammatory response may also contribute. We have previously shown that neonatal rats exposed to hyperoxia develop a pulmonary inflammatory response at a slower rate and to a lesser degree than adult rats (5).

In order for neutrophils to enter into a site of inflammation, they must migrate by a multistage process with initial loose attachment to the endothelium followed by firm adhesion. Adherence of neutrophils to the surface of the vascular endothelium is mediated by a series of adherence molecules including the selectins, the integrins, and their counterstructures. CD11b/CD18, a glycoprotein of the ß2-integrin family located on the surface of the neutrophil, is rapidly upregulated after activation and promotes strong attachment of the neutrophil to the vascular endothelium and neutrophil transmigration through the endothelium at a site of inflammation (6). L-selectin is a member of the selectin family of lectin-binding proteins, and is located on the cell surface of granulocytes, lymphocytes, and monocytes. It participates in the initial "loose" attachment of leukocytes to endothelium under the shear stress of flowing blood. In contrast to CD11b/CD18, L-selectin is rapidly shed from the cell surface by a proteolytic process after activation of the neutrophil (7). This process produces a chemically stable molecule, soluble L-selectin, which is the extracellular domain of membrane-bound L-selectin (8). Soluble L-selectin is thought to be able to inhibit leukocyte attachment to endothelium when it is present in high levels (8). Changes in soluble L-selectin may influence neutrophil–endothelial interactions in vivo and thereby modify the inflammatory response.

It has been demonstrated that the human neonatal neutrophil has both diminished adherence to activated endothelium and decreased directed migration to a chemotactic stimulus (9, 10). These defects in neutrophil function may contribute not only to defects in host defense and increased risk of infection, but also to protection from reactive oxygen intermediates produced by neutrophils. Studies in the human and rabbit neonate have demonstrated alterations from the adult in the neutrophil adherence proteins, CD11b/CD18 and L-selectin, which contribute to the defects in neutrophil function (9, 11, 12). To our knowledge, little is known about these adherence proteins in the neonatal rat. We hypothesized that similar differences from the adult would be noted on the neutrophils of the developing rat. Therefore, we examined L-selectin and CD18 on the neutrophil surface and soluble L-selectin in the serum of the neonatal rat at 0–24 and 72 h and at 1, 2, and 3 wk of age. Because of our previous findings of decreased neutrophil influx into the lung of the neonatal rat exposed to hyperoxia, we also examined the adherence proteins and soluble L-selectin in neonatal rats exposed to hyperoxia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Oxygen Exposures
Oxygen exposures were performed as previously described in our laboratory (5). Young adult male rats weighing 175–200 g (~ 44–48 d) were exposed to air or O2 for 56 h. Samples were obtained at 56 h in the adults because, in our experience, this time immediately precedes onset of oxygen-induced mortality in adult rats. Timed-gestation pregnant female Sprague-Dawley rats were obtained from Sasco, Inc. (Omaha, NE), and were allowed to spontaneously deliver at term. Neonates from two to four litters were pooled and divided between air and oxygen exposure groups. Because litters were pooled, oxygen exposure was delayed until all four dams used in each experiment had delivered. Deliveries occurred within 12–24 h, so all exposures were begun within the first 24 h of life. The animals were exposed to oxygen in airtight plastic cages with an oxygen flow rate of 5 liters/min. Measurement of oxygen concentration exiting the cages was >= 98%. Dams were switched between air and oxygen exposure cages every 24 h to limit oxygen toxicity. Neonatal exposures were continued for 7 d (a time at which significant mortality begins for oxygen-exposed pups). Samples from O2-exposed pups were obtained at 72 h and 7 d. Air exposures of the neonatal rats were continued for 21 d, and samples were obtained at 0–24 (pre-exposure) and 72 h and 7, 10, 14, and 21 d. Experiments were performed with the approval of the Animal Care and Use Committee of the University of Texas Medical Branch.

Blood Processing
Neonates were anesthetized with intraperitoneal sodium pentobarbital at a dose of 75 mg/kg body weight. Blood was obtained by intracardiac puncture from neonatal and adult rats and mixed with 100 U sodium heparin per ml blood. Aliquots of blood were lysed with 1% acetic acid, and total leukocytes and neutrophils were counted in a hemocytometer. An additional aliquot was centrifuged at 9,700 x g and the serum was frozen at -70°C for measurement of soluble L-selectin; the remaining blood was processed on ice for flow cytometry. Blood from four to five neonates at the 0–24-h time point and two neonates at the 72-h and 7-d time points was pooled for each sample. Thereafter blood from each individual neonate constituted a sample. Numbers for each group in air and O2 are given in the figure legends.

Flow Cytometry
Hybridomas for hamster anti-rat L-selectin monoclonal antibodies (HRL3 and HRL4) were a gift from Dr. Donald Anderson (Upjohn Laboratories, Kalamazoo, MI). Monoclonal antibody from supernatants of hybridoma cell cultures was purified over a protein A column (Pierce Chemical Co., Rockford, IL) and quantitated. For flow cytometry, 0.18 ml of whole blood was incubated with antibody for 20 min at 4°C. Hamster anti-rat L-selectin (HRL4) was used at a concentration of 15 µg/ml, and mouse anti-rat CD18 (MCA 775; Bioproducts of Science, Indianapolis, IN) at a concentration of 10 µg/ml. A saturation curve was determined for the L-selectin antibody using concentrations of 2.5–20 µg/ml. After a concentration of 10 µg/ml, no further increase in mean channel fluorescence was noted when increasing antibody concentrations were used. For L-selectin studies, the cells were then stained with fluorescein isothiocyanate–conjugated goat anti-hamster IgG (Cappel Research Products, Durham, NC) for 25 min at 4°C. For studies of CD18, a fluorescein isothiocyanate–conjugated goat anti-murine IgG (Accurate Chemical Corp., Westbury, NY) was employed. Subsequently, the cells were lysed for 8 min using FACS lysis buffer (Becton-Dickson, San Jose, CA) and then were washed in phosphate-buffered saline (PBS) and fixed in 1% paraformaldehyde.

For measurements of expression in activated neutrophils, N-formyl-methionyl-leucyl-phenylalanine (FMLP) was used as the activating agent. FMLP was added to aliquots of whole blood at a concentration of 10-7 M, incubated at 37°C for 30 min, and then rinsed with PBS and stained in the same manner as unstimulated samples. In studies of L-selectin expression on neutrophils of adult rats, FMLP activation did not result in reliable loss of L-selectin from rat neutrophils. The appearance on flow cytometry suggested aggregation of neutrophils after interaction with FMLP and the L-selectin antibody, which is a pentameric IgM antibody. However, CD18 consistently increased after FMLP, and studies were done of CD18 after FMLP activation in adults and neonates at 0–24 h, 72 h, and 7 d (n = 4 in each group).

Single-color flow cytometry was performed using a Becton-Dickinson FACScan (Becton-Dickinson, Mountain View, CA) as previously reported (13). Flow cytometry was performed on the day of staining because previous studies in our laboratory demonstrated a loss of L-selectin from rat neutrophils during storage after processing and fixation. An electronic gate was set on the neutrophil and non-neutrophil populations, based on forward-angle versus right-angle light scatter. All analyses were simultaneously run with an isotype control (mouse IgG1 for the CD18 and hamster IgG for the L-selectin antibodies). Analyses were conducted with the LYSIS II program. The log of fluorescence intensity (mean channel fluorescence) was compared between groups.

Enzyme-Linked Immunosorbent Assay for Soluble L-Selectin
Serum soluble L-selectin was measured using a modification of the sandwich enzyme-linked immunosorbent assay (ELISA) method for the HRL3 and HRL4 antibodies previously reported by Tamatani and coworkers (14). ELISA plates were incubated at 4°C overnight with primary antibody (HRL3) at a concentration of 20 µg/ml. After blocking with PBS/10% calf serum (CS), undiluted serum sample (0.1 ml) was then incubated on the plates for 4 h at room temperature and washed with PBS/2% CS. Secondary antibody (HRL4) was biotinylated using NHS-LC-Biotin II (Pierce Chemical Co., Rockford, IL) as per manufacturer's instructions. Unreacted biotin was removed using a BioRad desalting column and the antibody was then concentrated on a Centricon-30 microconcentrator (Amicon, Inc., Beverly, MA). Biotinylated HRL4 was added at a concentration of 3 µg/ml, and the plates were incubated for 1 h. After washing, the plates were incubated with avidin-conjugated horseradish peroxidase (0.1 ml at 1:800 dilution). Color was developed with o-phenylenediamine (OPD), and H2O2 and absorbance read at 492 nM. Controls consisted of similarly treated wells initially coated with hamster IgG at a concentration of 10 µg/ml instead of primary antibody. Results are expressed as absorbance of sample minus absorbance of IgG control.

Statistical Analysis
Results of mean channel fluorescence (for surface expression of L-selectin and CD18) and absorbance (for soluble L-selectin) are expressed as mean ± SD. Differences between neonates at each neonatal age and adults were analyzed by one-way ANOVA using Newman-Keuls correction for multiple comparisons or by Kruskall-Wallis testing with Dunn's test for multiple comparisons if the data were not normal in distribution. Differences between neutrophil counts and results after air and oxygen exposure were analyzed by a two-way ANOVA with Newman-Keuls testing for multiple comparisons. A P value of < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
There was no mortality of adult animals or of dams during the oxygen exposures. In our experience, switching of dams between oxygen and air exposure groups every 24 h ameliorates oxygen toxicity and allows survival of these dams for at least 14 d. There was minimal mortality of neonatal rats for the first 7 d. Thereafter, in the continuous >= 98% O2 exposure group, there was 67% mortality by 10 d. Therefore, the numbers in the 10 d O2 group were low and were not included in the results because they may not be completely representative of that group.

Table 1 shows neutrophil counts in the peripheral blood of air-exposed adults and neonates at various ages. Neutrophil counts are tabulated in the adults after 56 h of O2 exposure and in neonates after 72 h and 7 d of O2 exposure. The data for adults in air and O2 has been previously reported (15). Neutrophil counts in the air-exposed groups did not significantly differ between adults and neonates at any age. However, neutrophil counts tended to be higher in air-exposed neonates at 0–24 and 72 h compared with adults. In the air-exposed neonates, neutrophil counts dropped with age after 72 h. Compared with older neonates, counts in the 0–24- and 72-h-old neonates were significantly higher than in the 10 and 14–21 d neonates. After 56 h of O2 exposure, neutrophil counts increased by 6-fold in the adult rats. Neutrophils tended to increase in the neonates after O2 exposure but to a less marked degree than in the adults. The process was also delayed in the neonates. Neutrophils in the 72-h neonates did not differ between air and O2. After 7 d of O2 exposure, neutrophils were 2.5-fold higher than in the air-exposed neonates, but the difference was not significant due to variability in the counts after hyperoxia at this age.


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  		TABLE 1 Neutrophil counts in peripheral blood

 
Expression of Neutrophil Adhesion Molecules with Age
Figure 1 shows expression of L-selectin on neutrophils of adult and neonatal animals at the various ages (air exposure only). L-selectin expression did not differ between the adult and the 0–24-h neonate. L-selectin expression then dropped at 72 h to 60% of adult values and was 66% of adult values at 7 d. These values were significantly lower than the adult (P = 0.011). Thereafter, expression rose at 10–21 d and approximated that of the adult.



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  		Figure 1. Expression of L-selectin on neutrophils of developing rats. Mean channel fluorescence for L-selectin on neutrophils of adults and neonates at various ages after air exposure. Data are expressed as mean ± SD; *P < 0.05 for adult versus neonate; n = 9 for adult, n = 9 for 0–24 h, n = 5 for 72 h, n = 8 for 7 d, n = 4 for 10 d, n = 12 for 14 d, n = 4 for 21 d.

 
Figure 2 compares soluble L-selectin in the serum of adult and neonatal rats. Soluble L-selectin is expressed as ELISA absorbance values because there are no available quantitated amounts with which to construct a standard curve. Soluble L-selectin levels in 0–24- and 72-h rats were significantly higher than in adults. Thereafter, soluble L-selectin levels fell to adult values from 7–21 d of age.



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  		Figure 2. Levels of soluble L-selectin in the serum of developing rats. Soluble L-selectin, expressed as absorbance, in serum of adult and neonatal rats at various ages after air exposure. Data are expressed as mean ± SD; *P < 0.05 for adult versus neonate; n = 8 for adult, n = 11 for 0–24 h, n = 9 for 72 h, n = 4 for 7 d, n = 4 for 10 d, n = 4 for 14 d, n = 4 for 21 d.

 
Figure 3 shows expression of CD18 on unstimulated neutrophils at the various age groups. Expression of CD18 in the 0–24-h old neonates was significantly higher than in the adults (P < 0.001) and was also higher than neonates at all other ages. Expression from 72 h through 21 d did not significantly differ from the adult, although there was a trend toward higher values at all the neonatal time points.



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  		Figure 3. Expression of CD18 on neutrophils of developing rats. Mean channel fluorescence for CD18 on neutrophils of adults and neonates at various ages after air exposure. Data are expressed as mean ± SD; *P < 0.05 for adult versus neonate; n = 9 for adult, n = 14 for 0–24 h, n = 9 for 72 h, n = 12 for 12 d, n = 5 for 10 d, n = 11 for 14 d, n = 4 for 21 d.

 
Table 2 shows expression of CD18 after FMLP activation. Only data paired with FMLP were used, so the values for the unstimulated cells differ slightly from those of Figure 3. CD18 expression increased at all ages after FMLP activation of the neutrophil. Activated expression was actually highest in the 0–24-h neonate, and was significantly higher at 0–24 h than at other ages. CD18 expression in activated neutrophils did not differ between air- and O2-exposed animals, but there was a trend toward higher expression in the O2-exposed adults compared with air-exposed adults. When analyzed as the percentage increased from unstimulated values, upregulation was 50% lower in the 0–24- and 72-h neonates compared with the adult. There was also a 60% lower percentage increase in the adult after O2 exposure.


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  		TABLE 2 CD18 expression after FMLP stimulation

 
Effect of Oxygen Exposure on Expression of L-selectin and CD18
Figure 4A shows expression of L-selectin after exposure of adults to O2 for 56 h and in neonates exposed to O2 for 72 h and 7 d. The values for air exposure are identical to those in Figure 1. There were no significant differences between expression after air and O2 exposure in the adults or in the neonates at any age.





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  		Figure 4. Expression of L-selectin (A), levels of soluble L-selectin (B), and expression of CD18 (C) on neutrophils of air- and oxygen-exposed rats. Data were measured in adults and in neonatal rats aged 72 h and 7 d exposed to air (black bars) or O2 (gray bars). Data are expressed as mean ± SD; *P < 0.05 for air versus O2. Numbers in each group are as follows: L-selectin: n = 9 for adult air, n = 7 for adult O2, n = 5 for 72 h air, n = 7 for 72 h O2, n = 8 for 7 d air, n = 8 for 7 d O2; soluble L-selectin: n = 8 for adult air, n = 4 for adult O2, n = 9 for 72 h air, n = 9 for 72 h O2, n = 4 for 7 d air, n = 8 for 7 d O2; CD18: n = 9 for adult air, n = 7 for adult O2, n = 9 for 72 h air, n = 12 for 72 h O2, n = 12 for 7 d air, n = 14 for 7 d O2.

 
Figure 4B shows expression of soluble L-selectin after air and oxygen exposure. Soluble L-selectin did not significantly differ between the air and oxygen exposure groups in the adult or in the neonate, although there was a trend toward higher soluble L-selectin in the O2-exposed neonates at 7 d compared with air-exposed neonates.

Figure 4C shows CD18 expression in the two exposure groups. In the adult rat, there was a significantly higher CD18 expression after 56 h of O2 exposure (P = 0.008). In the neonates, CD18 did not differ between air and O2 exposure groups at either 72 h or 7 d.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we report the expression of L-selectin and CD18 on neutrophils of developing neonatal rats. Our data in the rat model agree with previous studies of L-selectin in other species in that L-selectin is lower in the neonatal rat compared with the adult. However, this diminished expression of L-selectin was not present until 72 h and persisted for only the first week of life. Previous studies in humans (11, 16, 17) and rabbits (12, 18) have shown lower expression of L-selectin in term neonates (< 24 h) compared with the adult. In the rabbit, Fortenberry and coworkers (12) showed that expression of L-selectin in the term rabbit was lower than the adult on Day 1, but reached adult levels by Day 2. We have previously shown that this lower expression of L-selectin in human neonates persists for at least 4 wk (19). The most likely explanation for the lower L-selectin noted on 3- and 7-d neonatal rat neutrophils is that of a developmentally-timed proteolytic process resulting in diminished surface expression of the molecule. The rat species is considered to be relatively immature compared with other species at term birth and the drop in rat L-selectin at 72 h instead of birth may reflect a developmental maturation similar to that of the human, but slightly delayed. The process is not likely to be due to the event of labor and/or delivery, because expression is not diminished at 0–24 h. All possible efforts were made to avoid neutrophil activation during the processing procedure, including rapid processing of samples, staining of whole blood without neutrophil separation procedures (17), and running the flow cytometry on the same day of processing. The findings of higher L-selectin at 0–24 h would make activation due to processing an unlikely explanation. It does not appear that the lower neonatal L-selectin is related to a deficiency in the ability of the neonatal neutrophil to produce and/or express the molecule on its surface, in view of higher expression at 0–24 h. In support of this, Smith and coworkers (20) demonstrated that L-selectin expression on human fetal neutrophils obtained by umbilical cord blood sampling is not different than that of the adult.

The lower L-selectin in the neonatal rat during the first week of life could account, at least in part, for the deficiency noted in neutrophil function of the neonate and for the delayed and diminished pulmonary recruitment of neutrophils in the neonate compared with the adult that we have previously demonstrated after hyperoxic exposure (5). Although surface expression of L-selectin was higher for the first 24 h, it was thereafter lower than that of the adult. Because neutrophil half-life is short, the higher levels at 24 h would not preclude a protective effect from O2 toxicity, which occurs over 7 d. Decreased L-selectin is associated with an impairment in neutrophil chemotaxis during shear stress and with decreased CD18-independent adhesion to endothelial cells in vitro (11). Several studies have shown the importance of L-selectin for neutrophil sequestration in the lung, but its role in oxygen toxicity is unknown. It has been demonstrated that L-selectin is responsible for slowing leukocyte transit and for leukocyte retention in pulmonary capillaries (21, 22). Other studies have demonstrated that certain types of lung injury that are dependent on neutrophils and mediated by oxygen radicals require L-selectin (23, 24). In such models, L-selectin–deficient animals have lower pulmonary toxicity. In hyperoxia-exposed rat lungs, blocking of L-selectin activity by fucoidin inhibited both arteriolar and venular rolling in an isolated perfused lung (25). We have shown in our laboratory that commercially available fucoidin may contain large quantities of endotoxin (F. C. Schmalstieg and colleagues, unpublished observations); therefore, this effect may also have been related to loss of L-selectin from the neutrophils. Of interest, Waisman and coworkers (26) demonstrated that administration of dexamethasone to preterm infants with BPD resulted in diminished expression of L-selectin on their neutrophils. This lower L-selectin could play a role in the anti-inflammatory effect of dexamethasone treatment for this disease.

We demonstrated a higher CD18 in the 0–24-h neonatal rat compared with the adult. Thereafter, unstimulated neonatal CD18 expression did not differ from the adult from 72 h through 21 d. Previous studies of neonatal CD11b/CD18 expression are conflicting. Our findings for CD18 in the 0–24-h neonate agree with those of Rebuck and colleagues (17) and Smith and coworkers (27), who demonstrated an elevated CD11b/CD18 on unstimulated term and preterm neonatal neutrophils. Other studies in humans (16, 28) and rabbits (12, 18) showed CD11b/CD18 either lower than, or similar to, that of the adult. Studies of human cord blood (16, 27, 28) and blood from neonatal rabbits (12, 18) have shown diminished upregulation of CD18 after activation in the neonate, and have correlated this with diminished stimulated adherence (28) and impaired transendothelial migration in neonatal neutrophils (9). Our data in the neonatal rat did not agree with these previous findings. Although we demonstrated a lower percentage of upregulation of CD18 in the 0–24- and 72-h neonates, the total expression of CD18 in activated neutrophils of the neonate was similar or higher than adults.

Changes in surface expression of L-selectin and CD11b/CD18 have been reported in acute systemic inflammatory states. In the neonate, L-selectin has been reported to be decreased and CD11b to be increased in cord blood of infants with acute bacterial sepsis (29, 30). Because hyperoxia results in a pulmonary inflammatory event, we hypothesized that release of mediators from inflamed endothelium or other blood cells might result in sufficient acute systemic inflammation to result in changes in expression of these surface proteins. Fifty-six hours of oxygen exposure resulted in significant elevation of CD18 expression in the adult rat. The percentage upregulation of CD18 after FMLP in these oxygen-exposed adults was also lower than the upregulation in air-exposed adult controls, suggesting activation of the neutrophil as a result of hyperoxic exposure. There were no differences in CD18 expression in neonatal rat neutrophils or in L-selectin expression in any age group as a result of hyperoxia.

Levels of soluble L-selectin in the air-exposed neonates were higher at 0–24 and 72 h, and thereafter did not differ from adult levels. These data conflict with those in the human, which have demonstrated lower levels of soluble L-selectin in the preterm and term human compared with the adult (17, 31). It has previously been postulated that levels of soluble L-selectin primarily reflect long-term neutrophil turnover and granulopoiesis rather than acute levels of expression or activation of circulating neutrophils (8, 17). Previous studies have demonstrated that activation of all circulating neutrophils in the human could not produce the amount of measured soluble L-selectin in human serum (8). Buhrer and colleagues (31) noted that soluble L-selectin levels in cord blood were correlated not only with gestational age, but also with absolute neutrophil counts. It has also been demonstrated that levels of soluble L-selectin correlate with neutrophil counts in patients with leukemia (32). Our findings of higher neutrophil counts in 0–24- and 72-h neonatal rats would support this hypothesis as an explanation for the higher soluble L-selectin levels at these ages. However, because soluble L-selectin can also bind to endothelium via ligands for L-selectin (8), we also hypothesize that measured levels of soluble L-selectin are determined not only by neutrophil numbers and turnover, but also by the expression of ligands for L-selectin. Given the extensive microvascular surface area, this could account for extensive binding and loss of circulating soluble L-selectin. Findings of low levels of soluble L-selectin in diffuse inflammatory states and findings showing that low levels predict progression to ARDS support this hypothesis (33). Also, we have previously shown that after LPS infusion, there is an acute drop in soluble L-selectin in the adult rat, supporting the hypothesis of an increase in ligand expression for L-selectin (15). It is possible that the ligand(s) for L-selectin in the very young neonate are not expressed to the same extent as in older neonates and adults, resulting in higher soluble L-selectin at 0–24 and 72 h. Unfortunately, because the exact ligand(s) for L-selectin is unknown, we were unable to test this hypothesis.

Soluble L-selectin tended to increase in neonates after 7 d of hyperoxia compared with air. Again, this may be a reflection of increased neutrophil production and turnover suggested by increased neutrophil counts, which were seen in the oxygen-exposed animals. The rise in soluble L-selectin in the neonate occurred only after 7 d of hyperoxia, a time at which neutrophil counts doubled. However, it is of interest that soluble L-selectin did not rise to such a great extent in the adult after O2 exposure (air versus O2, P = NS for adult), even though neutrophil counts in the adult rose to approximately six times the air levels. These findings would support our hypothesis that soluble L-selectin is determined by the interplay of neutrophil numbers and L-selectin ligand expression. It is possible that endothelial ligands for L-selectin are not upregulated in the neonate to as great an extent as in the adult after hyperoxic exposure. We have previously shown that E-selectin protein expression on pulmonary endothelium increases after 48 and 60 h of O2 exposure in the adult. Although it also increases in the neonate, it does not do so until after 7 d of hyperoxic exposure and to a lesser extent (34). Although E-selectin is not a ligand for L-selectin, it could be a marker for endothelial expression of other ligands.

It is postulated that soluble L-selectin serves an anti-inflammatory role. When activated, L-selectin is cleaved from the neutrophil by a proteolytic process. As previously mentioned, the resulting soluble L-selectin can bind to endothelial ligands and has been shown to competitively bind with intact L-selectin at the endothelial surface (8). Circulating soluble L-selectin may be functional in vivo as suggested by immunohistochemical studies revealing soluble L-selectin bound to endothelial cells (8). Soluble IgG L-selectin chimeras have been shown to protect from neutrophil-mediated lung injury (35). Therefore, the comparatively higher neonatal levels of soluble L-selectin in the early neonatal period and after neonatal oxygen exposure could be a further mechanism to protect the neonate from the inflammation associated with oxygen toxicity.

In summary, we report that, as in the neonatal human and rabbit, there are differences in expression of neutrophil adhesion molecules in the neonatal rat compared with the adult. L-selectin on neutrophils of the neonatal rat was lower than in the adult by 72 h and remained lower than the adult for 7 d. We also found that soluble L-selectin was higher in the air-exposed neonatal rat at 0–24 and 72 h, and tended to be higher after 7 d of hyperoxic exposure. Finally, we found that expression of CD18 in both unstimulated and activated neonatal neutrophils was similar to that in adults. These findings of no differences in CD18 expression between neonates and adults, even after activation, combined with our previous findings that pulmonary inflammation is a CD18-independent event in a model of O2 toxicity in the guinea pig (13), lead to the conclusion that deficient CD18 does not play a role in neonatal O2 tolerance. However, because neutrophil recruitment into the lung may be, at least in part, mediated by L-selectin, both lower surface L-selectin and higher soluble L-selectin might play a role in the diminished pulmonary inflammatory response in the neonatal rat that we have previously noted after hyperoxia. This mechanism could also protect the young animal from other types of inflammatory injury.


   Acknowledgments
 
This work was supported by a Child Health Research Center Grant CHD27841 from the National Institute of Child Health and Human Development. The hybridomas for hamster anti-rat L-selectin antibodies (HRL3 and HRL4) were a gift of Dr. Donald Anderson, Upjohn Laboratories, Kalamazoo, MI. The authors thank Ms. Kelli Noworyta for technical assistance, and Ms. Debbie Olsen and Ms. Carter Adcock for assistance with preparation of the manuscript.

Received in original form July 19, 2002

Received in final form April 16, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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