INTRODUCTION

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ENDOTOXIN [lipopolysaccharide (LPS)] exerts profound effects on the immune system that can lead to multiple-organ failure, septic shock, and even death (20). The intense activation of the immune system that accompanies LPS administration affects a variety of cells, including endothelial cells, macrophages, and circulating inflammatory cells, such as neutrophils and lymphocytes. Many of the responses of these cells to LPS are dependent on transcription-dependent synthesis of proteins that can act to either promote or protect against inflammatory cell-mediated tissue damage. For example, LPS is known to promote the production of cytokines by macrophages (5) as well as the upregulation of leukocyte-recruiting adhesion molecules on the surface of endothelial cells (27). However, LPS is also known to induce the biosynthesis of antioxidant enzymes [e.g., superoxide dismutase (SOD), catalase], heat shock proteins, as well as the inducible isoform of nitric oxide (NO) synthase (iNOS) (2).

The contention that some proteins produced by cells exposed to LPS are protective to the host and tend to diminish the deleterious consequences of LPS-induced immune activation is supported by the phenomenon of LPS-induced preconditioning (LPS-PC) or tolerance (13). The LPS-PC response represents a situation wherein exposure to low-dose LPS renders tissues resistant to subsequent challenge with a higher (even lethal) dose of LPS (26) or to a different major stress (e.g., ischemia-reperfusion). The protective effect of the initial LPS challenge is exemplified by decreased mortality, improved cardiac and lung function, decreased tissue infiltration by inflammatory leukocytes, and a diminished activity of macrophages after exposure to the second dose of LPS (32). Although LPS-PC has been demonstrated in several tissues, the mechanism underlying this induction of resistance to inflammatory stimuli, such as LPS and ischemia-reperfusion, remains poorly understood. Reactive oxygen metabolites and NO have been implicated as potential initiating factors in LPS-PC; however, the contribution of these factors to LPS-PC has not been rigorously tested in vivo. Furthermore, it remains unclear whether endothelial cells in vivo are as profoundly affected by LPS-PC as their inflammatory cell counterparts, particularly macrophages. Because LPS is known to elicit an increased production and expression of endothelial cell adhesion molecules, such as the selectins (11), it is conceivable that a blunted expression of these adhesion molecules accounts for at least part of the LPS-PC response. This possibility is supported by published studies dealing with the phenomenon of ischemic preconditioning, wherein inhibition of selectin-mediated leukocyte rolling appears to play an important role (1). Reports implicating both NO and reactive oxygen metabolites in ischemic preconditioning (19), coupled to the well-characterized influence of LPS on SOD and NOS activities (28), also raise the possibility that any effect of LPS-PC on selectin expression may be mediated through changes in NO and/or superoxide.

The overall objective of this study was to test the hypothesis that LPS-PC is associated with an increased resistance of vascular endothelial cells to activation by a subsequent challenge with LPS. To achieve this objective, the expression (protein) of E- and P-selectins was used as a marker of endothelial cell activation in the intestinal vasculature. These adhesion molecules contribute significantly to the recruitment of leukocytes in different inflammatory conditions. Hence, an LPS-induced downregulation of the endothelial selectins could explain the diminished neutrophil recruitment previously observed in lungs after LPS-PC (32). A second objective of this study was to apply different mutant mice to assess the possible contribution of NO, superoxide, and lymphocytes to LPS-PC-induced changes in the expression of E-selectin.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Monoclonal antibodies. The monoclonal antibodies (MAbs) used for in vivo assessment of E- and P-selectins expression were 10E9.6, a purified binding rat immunoglobulin (IgG2a) that is specific for mouse CD62E (E-selectin) (36); RB40.34, a purified binding rat immunoglobulin (IgG1) that is specific for mouse CD62P (P-selectin) (33); and P-23, an irrelevant, nonbinding murine IgG1 directed against human P-selectin (Pharmacia-Upjohn, Kalamazoo, MI) (21). The specific binding (10E9.6 or RB40.34) and nonbinding (P-23) MAbs were labeled with ¹²⁵I and ¹³¹I, respectively (Du Pont-New England Nuclear, Boston, MA), using the 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril (Iodo-Gen) method (14). Briefly, iodogen (Pierce, Rockford, IL) was dissolved in chloroform at a concentration of 0.5 mg/ml, and 250 µl of this solution were placed in glass tubes and evaporated under nitrogen. A 250-µg sample of MAb was added to each iodogen-coated tube, and either ¹²⁵I or ¹³¹I (1 µCi/µg protein) was added. The mixture was incubated on ice, with periodic stirring for 5-10 min. The total volume was brought to 2.5 ml by adding PBS (pH 7.4). Thereafter, the coupled MAb was separated from free ¹²⁵I or ¹³¹I by gel filtration on a Sephadex PD-10 column (Pharmacia, Uppsala, Sweden). The column was equilibrated (50 ml) and then eluted with PBS containing 1% bovine serum albumin. Four fractions were collected, the second of which contained the radiolabeled MAb. Absence of free ¹²⁵I or ¹³¹I was ensured by extensive dialysis of the protein-containing fraction. Less than 1% of the activity of the protein fraction was recovered from the dialysis fluid. Radiolabeled MAbs were stored at 4°C and used within 3 wk (¹³¹I-labeled MAb) or 2 mo (¹²⁵I-labeled 10E9.6 or ¹²⁵I-labeled RB40.34) after labeling.

Animals. Several strains of mice were used in this study. Six-to-eight week-old male wild-type C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME), weighing 27.6 ± 0.4 g (n = 22), were used to determine whether LPS-PC alters E-selectin expression induced by a second dose of LPS. In addition, the C57BL/6J mice were used as controls (background strain) for the studies performed using severe combined immunodeficient (SCID) mice (n = 34; 22.4 ± 0.2 g), both heterozygous positive (SOD-Tg) and negative (SOD-nonTg) Cu/Zn SOD transgenic [TgN(SOD1)3Cje] mice (n = 48; 26.9 ± 0.8 g) (12), and endothelial NOS (eNOS)-deficient (n = 17; 32.7 ± 3.8 g) mice (35). Because the iNOS-deficient mice (n = 41; 33.1 ± 0.6 g) were developed on a different background strain (B6×129) (22), additional control experiments were performed using these mice (n = 40; 29.7 ± 0.7 g). Another group of C57BL/6J mice (n = 23; 26.3 ± 0.5 g) was used to assess the influence of LPS-PC on P-selectin expression. Changes in plasma lactate concentration, circulating leukocyte counts, and tissue myeloperoxidase (MPO) activity (an index of neutrophil sequestration) (see MPO activity assay) were also assessed in a third group of C57BL/6J mice (n = 21; 24.3 ± 0.3 g).

Animal procedures. The mice were anesthetized subcutaneously with 150 mg/kg body wt of ketamine and 7.5 mg/kg body wt of xylazine. The right jugular vein and right carotid artery were cannulated with polyethylene tubing (PE-10). To measure E- or P-selectins expression, a mixture of 10 µg binding ¹²⁵I-labeled anti-E- or anti-P-selectin MAb and 0.5-5 µg of nonbinding ¹³¹I-MAb, adjusted to ensure a total ¹³¹I injected activity of 500,000 ± 100,000 counts/min, were injected through the jugular vein catheter (total volume 200 µl). Mice were then heparinized with 50 IU heparin sodium in 0.2 ml saline. Blood samples (200 µl) were obtained through the carotid artery catheter 5 min after injection of the MAb mixture for measurement of plasma ¹²⁵I and ¹³¹I activity (50 µl). Thereafter, an isovolemic blood exchange was rapidly performed with bicarbonate-buffered saline (6 ml) through the carotid artery catheter. The thoracic inferior vena cava was severed and flushed with bicarbonate-buffered saline (15 ml) through the carotid artery catheter. All the organs were harvested and weighed before radioactivity measurements. These experimental procedures were performed according to the criteria outlined by the National Institutes of Health and approved by the Louisiana State University Health Sciences Center-Shreveport, Committee on Animal Care and Use.

Calculation of E- and P-selectins expression. The method for calculation of E- and P-selectins expression has been previously described (11). In brief, specific binding ¹²⁵I-anti-E- or P-selectin MAb and nonspecific binding ¹³¹I-MAb in different organs and in 50-µl aliquots of cell-free plasma were counted in a 14800 Wizard 3 gamma counter (Wallac, Turku, Finland), with automatic correction for background activity and spillover. The injected activity in each experiment was calculated by counting a 2-µl sample of the mixture containing the radiolabeled MAbs. The radioactivities remaining in the tube used to mix the MAbs and the syringe used to inject the mixture were subtracted from the total calculated injected activity. The accumulated activity of E- or P-selectin MAb in an organ was expressed as nanograms of ¹²⁵I-anti-E- or P-selectin MAb per gram of tissue. E- or P-selectin expression was calculated by subtracting the accumulated activity of the irrelevant, nonbinding ¹³¹I MAb (¹³¹I-P-23) from the activity of the specific binding ¹²⁵I-anti-E- or P-selectin MAb (¹²⁵I-10E9.6 or ¹²⁵I- RB40.34, respectively). Previous studies have shown that MAbs retain their functional activity after radioiodination, as evidenced by a similar effectiveness of labeled and unlabeled MAbs to block leukocyte adherence in rat mesenteric venules (30). In addition, our laboratory showed that constitutive and endotoxin-induced expression of E-selectin is not detectable in the small intestine and other tissues of E-selectin-deficient mice, unlike their wild-type counterpart (11).

Experimental protocols. The dual-radiolabeled MAb technique was used to quantify the expression of E- and P-selectins in the intestine and other regional vascular beds of wild-type and mutant mice (see Calculation of E- and P-selectins expression) after LPS (Escherichia coli lipopolysaccharide, serotype 0111:B4, Sigma Chemical, St. Louis, MO) administration. Because previous studies (4) indicate that E-selectin expression peaks at 3 h after administration of LPS, E-selectin expression was assessed at 0 h (control) and 3 h after a dose of 0.5 µg after a priming dose of 1 µg LPS given 24 h earlier (LPS-PC). To determine whether the phenomenon of LPS-PC also affects LPS-induced P-selectin upregulation, the expression of P-selectin was assessed at 4 h after LPS administration. The time of LPS measurement was chosen on the basis of preliminary data suggesting that P-selectin peaks at 4 h after LPS administration.

Determination of plasma lactate. Lactate concentration in plasma was determined from the enzymatic conversion of lactate to pyruvate coupled to the reduction of NAD (Sigma Diagnostics, St. Louis, MO). The stoichiometric formation of NADH + H⁺ was monitored spectrophotometrically at 340 nm on a Gilford 240 spectrophotometer, with completion of the reaction ensured using hydrazine trapping of the formed pyruvate. Briefly, 100 µl of heparinized blood were collected and immediately deproteinized in a chilled centrifuge tube containing 100 µl of 10% trichloroacetic acid. The blood precipitate was vortex mixed for 30 s, kept on ice for 10 min, and then spun 10 min at 1,500 g; the supernatant was stored frozen at 78°C for further analysis. After a second centrifugation, 50-µl test samples were mixed in a cuvette containing 1 ml glycine-hydrazine buffer (0.6 mmol/l, pH 9.2 at 25°C) and 100 µl NAD (15 mM). After duplicate baseline readings, the reaction was started by adding 3 µl lactate dehydrogenase (1,000 U/ml) into the cuvette followed by three 10-min readings with the reaction completed in 20 min at 25°C. The blank cuvette was prepared substituting 50 µl of 10% trichloroacetic acid for the sample. A standard cuvette contained 50 µl lactate standard (2.2 mM), with the reaction being linear over the concentration range of 0.1-4.4 mM. Sample lactate concentrations were calculated after subtracting the appropriate blanks, using the 2.2 mM lactate standard.

Blood leukocyte count. The number of circulating leukocytes was determined from a 25-µl blood sample obtained from the carotid artery. Leukocytes were stained by mixing the blood sample with 465 µl of 3% acetic acid and 10 µl of 1% crystal violet. Polymorphonuclear leukocytes and mononuclear cells were counted with the aid of a Neubauer hematocytometer.

MPO activity assay. MPO activity, which is widely used to quantify neutrophil accumulation in tissues, was assessed by using the o-dianisidine method (37). Lungs and small bowel were harvested and immediately stored frozen (78°C). The tissue samples were thawed, weighed, suspended (10% wt/vol) in 50 mM potassium phosphate buffer (KP_i), pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide buffer (0.1 g/20 ml KP_i), and homogenized. One milliliter of the homogenate was sonicated three times for 10 s and microcentrifugated at 12,000 rpm for 10 min at 4°C. The reaction was started by mixing and incubating the supernatant (100 µl) at 20-25°C for 5 min with a solution composed of 2,900 µl of 50 mM KP_i, 30 µl of 20 mg/ml o-dianisidine dihydrochloride, and 30 µl of 20 mM hydrogen peroxide. The reaction was stopped by adding 30 µl of 2% sodium azide. The change in absorbency was read at 460 nm at 5 min in a spectrophotometer (Hitachi U-2000, Hitachi Instruments, Dallas, TX), and MPO activity was expressed as the amount of enzyme necessary to produce a change in absorbency of 1.0 · OD unit · min¹ · g wet wt tissue¹.

Statistics. The data were analyzed using a one-way ANOVA with Scheffé's (post hoc) test (StatView 4.02 for Macintosh computers). All values are reported as means ± SE. Statistical significance was set at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Figure 1 summarizes the changes in mean arterial pressure (A), plasma lactate (B), blood leukocyte count (C), and myeloperoxidase activity in the lungs (D) in wild-type (C57BL/6) mice. Although there was a tendency for increased arterial pressure 3 h after 0.5 µg LPS, this change was not statistically significant. Blood pressure after a priming dose of LPS (LPS-PC) was not significantly different from the control (untreated) groups. Whereas plasma lactate was not significantly altered at 3 h after injection of 0.5 µg LPS, a significant increase in lactate was noted 3 h after 0.5 µg LPS, in those mice receiving a priming dose of 1.0 µg of LPS given 24 h earlier. Within 3 h after administration of 0.5 µg LPS, a significant leukopenia was noted. This leukopenic response to 0.5 µg LPS was observed whether or not the mice received the priming dose of LPS. MPO activity was increased in the lungs, but not in the small bowel, after 0.5 µg LPS administration, especially after priming with 1.0 µg LPS.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Mean arterial pressure (A), plasma lactate level (B), leukocytes count (C), and myeloperoxidase (MPO) activity in the lungs (D) without lipopolysaccharide (LPS; control), at 3 h after 0.5 µg of LPS ip (LPS0.5), and 3 h after 0.5 µg LPS in wild-type (C57BL/6J) mice receiving a priming dose of 1.0 µg LPS 24 h earlier (LPS-PC). PMNs, polymorphonuclear leukocytes; ns, no statistical difference. * P < 0.05 vs. control;  P < 0.05 vs. LPS0.5 (ANOVA).

Figure 2 illustrates the effects of LPS-PC on the E- and P-selectins responses of the intestinal vasculature to a subsequent LPS dose. Figure 2A illustrates that wild-type mice receiving a 0.5-µg dose of LPS produced an eightfold increase in E-selectin expression at 3 h. However, if the same protocol was applied to mice receiving the priming dose of LPS, a significant upregulation of E-selectin was not observed. Figure 2B illustrates that the phenomenon of LPS-PC could also be demonstrated for P-selectin; i.e., LPS-induced P-selectin expression is significantly attenuated by 24-h pretreatment with LPS. However, the magnitude of the inhibitory effect of LPS-PC was much greater for E-selectin (66%) than P-selectin (33%).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   E-selectin (A) and P-selectin (B) expression in the small bowel measured without LPS (control), at 3 h after 0.5 µg of LPS ip (LPS0.5), and 3 h after 0.5 µg LPS in wild-type (C57BL/6J) mice receiving a priming dose of 1.0 µg LPS 24 h earlier (LPS-PC). MAb, monoclonal antibody. * P < 0.05 vs. control;  P < 0.05 vs. LPS0.5 (ANOVA).

Figure 3 summarizes the E-selectin responses to LPS-PC in different mutant mice, all of which were developed on a C57BL/6 (wild-type) background. The pattern of responses to LPS (± LPS pretreatment) in the intestinal vasculature of SCID mice was very similar to that observed in wild-type mice. However, the preconditioning response was not as intense in the lymphocyte-deficient SCID mice compared with their wild-type counterparts, suggesting that lymphocytes contribute to the preconditioning response. The Cu/Zn SOD overexpressing mice (SOD-Tg), as well as their nontransgenic littermates (SOD-nonTg), exhibited a pattern of responses to LPS (± LPS pretreatment) that was essentially identical to that observed in the intestinal vasculature of wild-type mice.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   E-selectin expression in the small bowel measured without LPS (control), at 3 h after 0.5 µg of LPS ip. (LPS0.5), and 3 h after 0.5 µg LPS in wild-type (C57BL/6J) mice receiving a priming dose of 1.0 µg LPS 24 h earlier (LPS-PC) in severe combined immunodeficient (SCID) mice and superoxide dismutase (SOD) transgenic (SOD-Tg) mice compared with their wild-type counterparts (C57BL/6J and SOD nonTg, respectively). * P < 0.05 vs. control;  P < 0.05 vs. LPS0.5 (ANOVA).

Figure 4 presents the E-selectin responses to LPS preconditioning in mice that are genetically deficient in either eNOS or iNOS. Because the iNOS-deficient mice were developed on a B6×129 background, these wild-type mice were also studied. The E-selectin responses to LPS (± LPS pretreatment) in both groups of NOS-deficient mice did not differ from the responses observed in the intestine of their respective wild-type counterparts, indicating that neither isoform of the NO-producing enzyme contributes to the preconditioning response.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   E-selectin expression in the small bowel measured without LPS (control), at 3 h after 0.5 µg of LPS ip (LPS0.5), and 3 h after 0.5 µg LPS in knockout mice [endothelial nitric oxide synthase deficient (eNOS-/-) and inducible nitric oxide synthase deficient (iNOS-/-)] receiving a priming dose of 1.0 µg LPS 24 h earlier (LPS-PC), compared with their wild-type counterparts (B6×129, for iNOS only). * P < 0.05 vs. control;  P < 0.05 vs. LPS0.5.

In the other organs studied, such as lungs or heart (Table 1), LPS-PC was not demonstrated for E-selectin. However, in these organs, P-selectin was significantly decreased with 0.5 µg of LPS, after a priming dose given 24 h earlier.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Late preconditioning or tolerance is a well-recognized phenomenon, which differs from early preconditioning (25). Tolerance develops after exposure to sublethal doses of LPS and is characterized by attenuated responses to a subsequent LPS challenge, which can be manifested as reductions in the production of proinflammatory cytokines [e.g., tumor necrosis factor- (TNF-)], pyrogenesis, and mortality (26). In the heart, resistance to LPS and ischemia develops as early as 24 h after the initial exposure to LPS and persists for up to 7 days. It has also been shown that LPS-PC is abolished by protein synthesis inhibition. Of the proteins that have been implicated in LPS-PC, antioxidant enzymes (15, 24) and NOS (8) appear as likely candidates for the mediator(s) of this response. Recently published studies on the mesenteric microcirculation indicate that ischemic preconditioning is associated with reductions in free radical formation and enhanced NO production, which prevent P-selectin expression and the subsequent rolling, adhesion, and emigration of leukocytes in postcapillary venules (19).

In the present study, we addressed the possibility that LPS can confer tolerance of E- and P-selectins expression in the intestinal vasculature to a subsequent dose of LPS. In addition, we assessed the potential contributions of an antioxidant enzyme (SOD) and NOS to the LPS-PC-mediated changes in E-selectin expression. We observed that a priming dose of LPS attenuated the responses of E- and P-selectins expression in the gut microvasculature to a subsequent dose of LPS by 66 and 33%, respectively. These blunted inflammatory responses to the second dose of LPS were associated with an unaltered LPS-induced leukopenic response and leukocyte accumulation in the lungs. This suggests that, whereas the intestinal vasculature exhibits a diminished capacity to recruit leukocytes (because of the lower selectin expression) after LPS-PC, the ability of the lungs to entrap leukocytes after LPS is unaffected or even enhanced by LPS-PC. It is also noteworthy that our finding of a 33% reduction in LPS-induced P-selectin expression after LPS-PC compares with an 80% reduction in P-selectin expression in the rat intestine after ischemic preconditioning (10). Our observation that intestinal E-selectin expression is inhibited to a greater degree than that of P-selectin after LPS-PC is consistent with reports describing different mechanisms that regulate the transcription of these two endothelial cell adhesion molecules (17).

It is now well recognized that a variety of stimuli that promote endothelial cells to produce (and express) E-selectin exerts their genetic action through nuclear transcription factor-B (NF-B) (9). The regulatory element of the E-selectin gene has three binding sites for NF-B. Agents such as TNF-, LPS, and reactive oxygen metabolites are known to promote cytosolic activation of NF-B and its subsequent translocation into the nucleus, where it can activate the gene for E-selectin and other adhesion molecules (34). NO (23) and SOD (31) are two physiologically relevant compounds that have been shown to attenuate the activation of NF-B- cultured endothelial cells that is induced by LPS. These observations suggest that LPS-PC could result from an increased production of SOD and/or NO that is induced by the priming dose of LPS. These superoxide scavenging agents could then act to minimize the activation and nuclear translocation of NF-B that is normally elicited by the second dose (24 h later) of LPS.

To assess the possible contribution of SOD and/or NO to the LPS-PC-induced attenuation of LPS-mediated E-selectin expression, we capitalized on the availability of mutant mice that overexpress Cu/Zn SOD as well as mice that are genetically deficient in either eNOS (constitutive isoform) or iNOS. Our studies demonstrate that the increased E-selectin expression induced by a second dose of LPS (24 h after the priming dose of LPS) in the intestinal vasculature of these mutant mice was no different from the response normally observed in relevant wild-type mice. These findings suggest that the balance between superoxide and NO production is not a critical determinant of the LPS-PC-induced inhibition of E-selectin expression in the intestinal vasculature. Nonetheless, our results do not exclude the possibility that other reactive oxygen metabolites (e.g., hydrogen peroxide) or NO derived from neuronal NOS contribute to the LPS-PC responses of E-selectin.

Lactate is released in large quantities from sites of sepsis and inflammation (16) and after LPS administration in experimental animals (29). Therefore, lactate is often considered both as a marker of tissue hypoxia and as a product of inflammation. In this study, the acid-base imbalance that accompanies the altered E- or P-selectin expression suggests that cellular acidosis may contribute to blunted LPS-induced selectin expression. This possibility appears unlikely because lactate has been shown to promote the activation of NF-B, possibly through inhibition of the NF-B proteasomal degradation pathway (38, 39). Furthermore, it has been reported that transient exposure to lactate at levels detected after brief ischemic periods does not precondition the rat myocardium (3).

Although the effects were not dramatic, we did observe an attenuation of the LPS-PC response of E-selectin in mice that are genetically deficient in lymphocytes, i.e., SCID mice. Our laboratory previously reported that SCID mice exhibit a significantly blunted E-selectin response in the intestinal microvasculature after systemic administration of TNF- and that the blunted responses could be restored to wild-type levels when the SCID mice were reconstituted with T lymphocytes (18). These observations led us to conclude that T lymphocytes play an important role in amplifying the inflammatory responses to exogenous cytokines largely because the T cells respond to such a stimulus by producing more TNF- and other cytokines, which produce a larger increment in E-selectin expression. Such a mechanism could also explain why the absence of lymphocytes in SCID mice would result in an attenuated LPS-PC response, particularly if lymphocytes contributed to the production of TNF- or other cytokines that mediate the E-selectin response to the second dose of LPS. Support for such a mechanism is supported by published evidence that implicates TNF- in LPS-PC (6, 13).

Perspectives

An interesting feature of our findings is that, whereas LPS-PC is associated with a profound reduction in the expression of both endothelial cell selectins in the gut vasculature, this anti-inflammatory response was not associated with a corresponding reduction in the accumulation of inflammatory cells (neutrophils). This observation suggests that the selectins may not contribute significantly to the recruitment of neutrophils in the septic intestinal vasculature and that other mechanisms participate in this recruitment response. One possibility is that other adhesion molecules (e.g., intracellular adhesion molecule-1) are insensitive to (or enhanced by) LPS-PC and that these alternate adhesion molecules play a more dominant role in controlling the traffic of leukocytes under the septic conditions simulated in our experimental protocols. Alternatively, the sequestration of leukocytes after LPS-PC may reflect a passive response due to diminished leukocyte deformability (increased stiffness). Although such steric hindrance of leukocytes has been well characterized for the lung (7), it remains unclear whether LPS can induce such a response in peripheral vascular beds.