Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis

Cameron W. Lush, Gediminas Cepinskas, William J. Sibbald, and Peter R. Kvietys

Department of Physiology, University of Western Ontario, London, N6A 5C1; and Vascular Biology Program, Lawson Health Research Institute, London, Ontario, Canada, N6A 4G5


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)-/- mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS-/- and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS-/- mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS-/- mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS-/- mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS-/- mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.

neutrophils; cecal ligation and perforation; myeloperoxidase activity; intracellular adhesion molecule-2


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE RECRUITMENT OF LEUKOCYTES to the endothelial cell surface of the microvasculature, and subsequent infiltration into the interstitium, is believed to be the initial event that leads to multiple organ inflammation and dysfunction during sepsis (16, 24). This process involves a series of well-coordinated adhesive interactions mediated by adhesion glycoproteins expressed on leukocytes and the endothelium. The tethering and rolling of neutrophils (PMN) along the endothelium are regulated by the selectins (L-selectin on PMN and P- and E-selectin on endothelium), whereas firm adhesion is mediated primarily by intercellular adhesion molecule (ICAM)-1/beta 2-integrin interactions (24). Once firmly adherent to the endothelium, the PMN flatten, extend pseudopodia between endothelial cells, and emigrate into the interstitium. These invading PMN are believed to cause damage to the tissue (23).

PMN rolling along the endothelium is a prerequisite for subsequent adhesion and emigration. The endothelial cell selectins are not constitutively expressed on the cell surface. P-selectin, which is stored in the Weible-Palade bodies of endothelial cells, is rapidly expressed (within minutes) on the endothelial cell surface after stimulation (e.g., histamine, oxidants). There is also a transcription-dependent mechanism that can upregulate the expression of P-selectin (8, 9). It is believed that the stored P-selectin is responsible for the early recruitment of leukocytes, whereas the transcription-dependent expression of P-selectin is involved in the leukocyte rolling that is observed several hours after the onset of inflammation (8, 9). Although there is no preformed pool of E-selectin, endotoxin [e.g., lipopolysaccharide (LPS)] and cytokines [e.g., tumor necrosis factor-alpha and interleukin-1beta ] are capable of stimulating the synthesis and expression of E-selectin on the surface of endothelial cells (10, 15). The critical role of selectins in inflammation is evidenced by studies that show that interfering with selectin-mediated rolling of PMN prevents PMN infiltration and organ dysfunction (3, 4, 13, 17, 22).

Nitric oxide (NO), synthesized by NO synthase (NOS), is believed to modulate leukocyte-endothelial cell interactions by acting as an endogenous antiadhesive molecule (12). In vitro studies indicate that NO can attenuate endothelial cell adhesion molecule expression [E-selectin, ICAM-1, and vascular cell adhesion molecule-1] (6, 27, 28). The functional result of this inhibition is reduced PMN adhesion to endothelial cells (5, 11, 21). The constitutive isoform of NOS (cNOS) is continually expressed and produces small fluxes of NO, whereas the inducible NOS (iNOS) is a high-output isoform. In general, during sepsis, the activity of cNOS is decreased and iNOS activity is increased, resulting in substantially elevated levels of NO production (16). In the present study, we assessed the role of NO (derived from iNOS) in the expression of E- and P-selectin in mice exposed to sepsis. Our approach to address this issue involved the use of iNOS-deficient (iNOS-/-) mice.

We have recently reported that the induction of peritonitis in mice by cecal ligation and perforation (CLP) results in symptoms that closely mimic those observed clinically (hypotension, neutropenia, elevated blood lactate). In addition, CLP-induced peritonitis in these animals leads to increased expression of endothelial selectins in various organs (1). The purpose of the present study was to use our acute model of murine polymicrobial sepsis to determine the role of iNOS in the early (within 6 h) upregulation of the selectins on endothelial cells. As such, this is the first study to quantify the level of expression of E- and P-selectin in iNOS-/- mice following a septic insult. In addition, we present evidence that NO derived from iNOS contributes to selectin expression on the vascular endothelium during sepsis.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Animal protocol. Male C57/BL6 mice and iNOS-/- mice (C57/BL6 background) weighing 20-30 g were obtained from Charles River Canada (St. Constant, PQ) and Jackson Laboratories (Bar Harbor, ME), respectively. Sepsis was induced in mice using the CLP model as previously described (1, 20). Briefly, animals were anesthetized with 150 mg/kg body wt of ketamine and 7.5 mg/kg sc of xylazine. A midline incision ~2 cm long was made in the abdomen to expose the cecum and adjoining intestine. With the use of 2-0 silk, a ligature was placed around the cecum immediately distal to the ileocecal valve. The cecum was then opened by making a 5-mm incision at the antimesenteric border. The laparotomy was then closed using 4-0 silk, and the animal was given a 1 ml subcutaneous injection of saline for fluid resuscitation. Sham-operated mice underwent laparotomy but no ligation or cecal perforation. Control animals did not undergo surgery.

Two sets of animals were used in the present study. One set was used for measurements of systemic variables, plasma metabolites of NO, and tissue myeloperoxidase (MPO) activity. Another set was used for the in vivo determination of vascular E- and P-selectin expression.

Systemic variables. Six hours after induction of CLP, mean arterial pressure (MAP) was measured from a carotid arterial catheter that was connected to a pressure transducer and recorded with a multichannel amplifier recording system (Hewlett Packard 78353A). Blood samples were taken for analysis of both arterial blood levels of lactate and metabolic end products of NO (NOx-).

NOx- chemiluminescence detection. The metabolic end products of NO production (NO2- and NO3-; NOx-) were determined (29) in plasma samples using chemiluminescence detection in a Sievers Model 270 B analyzer (Sievers Instruments, Boulder, CO) with a Shimadzu Chromatopac C-R1A integrator (Shimadzu, Kyoto, Japan). Briefly, an aliquot of plasma was injected into a glass purge vessel containing a saturated solution of vanadium (III) chloride in hydrochloric acid (1 M) at 90°C, resulting in reduction of both NO2- and NO3- to NO. This gas-phase NO is carried by a continuous stream of inert gas (helium) from the purge vessel into an ozone-containing reaction chamber in the NO analyzer. The resultant chemiluminescent reaction between NO and ozone is detected by a photomultiplier tube, yielding an electric signal (mV) that was analyzed for area under the curve (AUC) calculations on the chromatographic integrator. The analyzer was calibrated daily and rechecked periodically during analysis of samples, which were referenced to a standard curve of AUC (mV × s) vs. NO3- concentration (50 nM-500 µM; r2 > 0.999).

Adhesion molecule expression. E- and P-selectin expression on the vascular endothelium of various organs was determined by using the dual radiolabeled antibody technique (1, 2, 9). The monoclonal antibodies (MAbs) used for the in vivo assessment of E-selectin expression were 10E9.6 (PharMingen), a binding rat IgG2a against mouse E-selectin and R35-95 (PharMingen), and a nonbinding purified rat IgG2a,kappa isotype-matched negative control. The P-selectin MAbs used were RB40.34 (PharMingen), a binding rat IgGg1 against mouse P-selectin, and the same nonbinding rat IgG2a,kappa described above. The use of both binding and nonbinding MAbs simultaneously allows for the correction of any nonspecific accumulation of the binding MAbs in a given tissue. The determination of ICAM-2 expression was made using a purified rat anti-mouse CD102 MAb (3C4; PharMingen).

With the use of the IODO-GEN (Pierce) method (2), all binding MAbs (10E9.6, RB40.34, and 3C4) were labeled with 125I, whereas nonbinding MAbs were labeled with 131I. Briefly, 200 µl of IODO-GEN (0.5 mg/ml in chloroform) were placed in a glass tube and evaporated under nitrogen. A 500-µg sample of MAb was incubated with 500 µCi of Na125I (or Na131I) in the IODO-GEN-coated tube on ice for 10 min. The total volume was then adjusted to 2.5 ml with PBS. The radiolabeled MAb was then separated from free 125I by gel filtration on a Sephadex PD-10 column (Pharmacia). PBS with 1% BSA was used to equilibrate the column and elute the radiolabeled antibody. Two 2.5-ml fractions were collected, with the second fraction containing the labeled MAb. Radiolabeled MAbs were stored at 4°C.

Six hours after CLP, the mice were reanesthetized with ketamine (150 mg/kg body wt sc) and xylazine (7.5 mg/kg body wt sc). The right jugular vein and right carotid artery were cannulated with polyethylene tubing (PE-10). A mixture of 10 µg of 125I-labeled binding MAb and an amount of 131I-labeled nonbinding MAb necessary to ensure a total activity of 500,000 ± 100,000 cpm (0.5-5 µg) was injected through the jugular vein catheter (total volume 200 µl). This dose was selected on the basis of previous studies demonstrating optimum activity and receptor saturation in the tissues examined under stimulated and unstimulated conditions (2, 9). Five minutes after injection of the MAb mixture, a blood sample (200 µl) was obtained through the carotid artery catheter for determination of plasma 125I and 131I activity (per 50 µl). Isovolemic blood exchange was rapidly performed with bicarbonate-buffered saline by infusion through the jugular vein and simultaneous withdrawal of blood/buffer from the aorta. The thoracic vena cava was severed and flushed with bicarbonate-buffered saline through the carotid artery. Various organs were harvested and weighed for quantification of P- or E-selectin or ICAM-2 expression.

A 14800 Wizard 3 gamma counter (Wallac) was used to count 125I and 131I activities in each organ and in a 50-µl plasma sample. The total injected activity in each experiment was calculated by counting a 2-µl sample of the preinjected mixture of radiolabeled MAb (less the radioactivity remaining in the tube used to mix the MAb and the syringe used to inject the mixture). P- or E-selectin expression was determined by subtracting the accumulated activity of the nonbinding 131I-MAb from that of the binding 125I-MAb activity in an organ and was expressed as nanograms of 125I-anti-E- or P-selectin MAb per gram of tissue.

MPO activity. Tissue samples were weighed, frozen, and analyzed for determination of MPO activity as previously described (2). MPO activity is commonly used as a measure of PMN accumulation in tissues. Briefly, samples were thawed, suspended (10% wt/vol) in potassium phosphate buffer (KPi; 50 mM, pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma), and homogenized. One milliliter of homogenate was sonicated three times and spun at 20,000 g for 10 min (4°C). The reaction was started by incubating 100 µl of supernatant with O-dianisidine solution (30 µl of 20 mg/ml O-dianisidine, 2,900 µl of 50 mM KPi, 30 µl of 20 mM H2O2) for 5 min at 25°C. The reaction was stopped by adding 30 µl of 2% sodium azide. The change in absorbency was read after 5 min at 450 nm using a microplate reader (Bio-Rad Model 3550-UV). MPO activity was expressed as the amount of enzyme necessary to produce a change in absorbency of 1.0 · min-1 · g wet wt-1.

Statistics. Comparison between genotypes (wild-type vs. iNOS-/-) of mice after CLP was made using one-way ANOVA followed by Student's t-test (with Bonferroni correction for multiple comparisons) and within a given genotype using a two-tailed t-test. Statistical significance for all tests was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

As shown in Table 1, 6 h after CLP there was a significant reduction in MAP compared with sham animals for both wild-type and iNOS-/- mice. MAP was reduced to similar levels in both wild-type and iNOS-/- mice. Furthermore, animals subjected to CLP also had an elevated level of circulating lactate, which is indicative of the development of sepsis. These changes in systemic variables are consistent with our previous studies using a similar model of sepsis in mice (1). All animals survived the 6-h CLP procedure.

                              
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Table 1.   Systemic variables of wild-type and iNOS-/- mice 6 h after CLP

The induction of CLP in wild-type mice resulted in a significant elevation in plasma NOx- levels, a measure of the metabolic end products of NO production (Fig. 1). In contrast, there were negligible levels of NO2- and NO3- in both CLP and sham-operated iNOS-/- mice.


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Fig. 1.   Plasma NO2- and NO3- (NOx-) levels of control, sham-operated, and cecal-ligated and -perforated (CLP) mice [wild type and inducible nitric oxide synthase (iNOS) deficient (-/-)]. Values are means ± SE (n = 5 in each group). # P < 0.05 vs. control; dagger  P < 0.05 between CLP wild-type and CLP iNOS-/- mice.

Expression of E-selectin 6 h after CLP was significantly increased in organs within the peritoneum (i.e., small and large bowel, pancreas) and an organ outside of the peritoneum (i.e., lung) in wild-type animals (Fig. 2). In iNOS-/- mice, however, there was no increase in the expression of E-selectin in these organs after CLP compared with sham. The data for other organs examined are summarized in Table 2. In general, similar trends were noted in other organs within the abdominal cavity.


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Fig. 2.   E-selectin expression in the small bowel (A), large bowel (B), pancreas (C), and lung (D) 6 h after CLP in wild-type and iNOS-/- mice. Values are means ± SE (n = 5-9 in each group). # P < 0.05 vs. control; * P < 0.05 vs. sham-operated; dagger  P < 0.05 between CLP wild-type and CLP iNOS-/- mice.


                              
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Table 2.   E-selectin expression in wild-type and iNOS-/- mice 6 h after CLP

In terms of P-selectin expression, variable responses were noted for both wild-type and iNOS-/- mice. Significant increases in P-selectin were noted 6 h after CLP in wild-type mice in organs within the abdominal cavity (small and large bowel, pancreas), but not in the lung (Fig. 3). There were significant decreases in the level of expression of P-selectin in both the large bowel and pancreas of iNOS-/- mice following CLP (Fig. 3). The data for other organs examined are summarized in Table 3. In general, similar results were noted for organs in the abdominal cavity.


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Fig. 3.   P-selectin expression in the small bowel (A), large bowel (B), pancreas (C), and lung (D) 6 h after CLP in wild-type and iNOS-/- mice. Values are means ± SE (n = 5-9 in each group). # P < 0.05 vs. control; * P < 0.05 vs. sham; dagger  P < 0.05 between CLP wild-type and CLP iNOS-/- mice.


                              
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Table 3.   P-selectin expression in wild-type and iNOS-/- mice 6 h after CLP

ICAM-2 expression in CLP wild-type and iNOS-deficient mice was assessed to determine tissue perfusion (Table 4). It has previously been reported that ICAM-2 is constitutively expressed on murine endothelial cells, and expression is not increased by stimulation with inflammatory cytokines (14, 31). Consistent with these findings, it is evident from Table 4 that, in general, there were no major changes in the expression of ICAM-2 after CLP in both wild-type and iNOS-deficient mice. These findings would indicate that, despite the hypotensive state of the septic animals, tissue perfusion was adequate for quantification of adhesion molecule expression.

                              
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Table 4.   ICAM-2 expression in wild-type and iNOS-/- mice 6 h after CLP

To determine whether these variations in the level of expression of E- and P-selectin correlated with changes in the accumulation of leukocytes in these organs, MPO activity in representative organs, i.e., the small bowel and lung, was assessed. As shown in Fig. 4, after the induction of CLP, MPO activity was increased in the small bowel of wild-type mice compared with sham animals. In contrast, induction of peritonitis in iNOS-/- mice did not result in an increase in MPO activity in the small bowel. CLP induced significant increases in lung MPO activity in both wild-type and iNOS-/- mice.


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Fig. 4.   Myeloperoxidase (MPO) activity in the small bowel (A) and lung (B) 6 h after CLP in wild-type and iNOS-/- mice. Values are means ± SE (n = 5 in each group). * P < 0.05 vs. sham.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of the present study was to assess the role of iNOS activity in the in vivo regulation of selectin expression after induction of sepsis (CLP) by using iNOS-/- mice. To confirm that these mice had not compensated for the lack of the iNOS gene, possibly by increased cNOS activity, we measured plasma NOx- levels (Fig. 1). Six hours after CLP, NOx- levels were significantly elevated in wild-type mice, indicative of increased iNOS activity and subsequent NO production. More importantly, our data indicate that there was no compensation for the lack of iNOS activity, because NOx- levels in the iNOS-/- mice were well below those of the wild-type mice, an observation consistent with previous studies (11, 18).

As previously reported (1), we show that 6 h after of CLP, E- and P-selectin expression is elevated within the microvasculature of organs in the abdominal cavity of wild-type mice. This CLP-induced increase in selectin expression is substantially reduced in iNOS-/- mice (Figs. 2 and 3). Moreover, this reduced expression corresponds to a reduced level of PMN recruitment in a representative organ, i.e., small bowel (Fig. 4). Together, these observations provide a compelling argument against the currently held view that NO inhibits adhesion molecule expression and PMN infiltration.

In apparent contrast to our observations, previous studies using iNOS-/- animals have shown more pronounced leukocyte-endothelial cell interactions (rolling) after endotoxin challenge (11). One possible explanation for these seemingly disparate results is that the previous study relied on LPS as the challenge, which may not reflect the complex situation that exists during polymicrobial sepsis. In support of this possibility are the results of a recent study in which we measured E- and P-selectin expression 6 h after induction of CLP in LPS-sensitive (C3Heb/FeJ) and LPS-insensitive (C3H/HeJ) mice (1). The CLP-induced increase in selectin expression was the same in both the LPS-sensitive and -insensitive mice. These findings indicate that bacteria-derived endotoxin (LPS) does not play an important role in the CLP-induced increase in selectin expression. Thus some degree of caution should be used in relating LPS-induced inflammatory responses to those induced by polymicrobial sepsis.

Our data indicate that NO production is important for the expression of E- and P-selectin after CLP. However, in vitro studies (6, 26-28) have found that NO can attenuate endothelial cell adhesion molecule expression. Two major differences between these latter studies and our present study exist that might offer an explanation for this discrepancy. First, the previous studies employed either NO donors or inhibitors to examine the effect of NO production on adhesion molecule expression in cultured human endothelial cells. In our study, we assessed the in vivo expression of selectins on endothelium of various organs in genetically altered mice. Thus it is conceivable that in situ endothelial cells of iNOS-/- animals respond differently from isolated cultured human cells. Second, adhesion molecule expression was induced in vitro by stimulation of endothelial cells with either interleukin-1 or tumor necrosis factor-alpha . This approach may not be representative of the in vivo situation, in which multiple factors are involved in endothelial cell stimulation during polymicrobial sepsis. Irrespective of the explanation for these seemingly dichotomous observations, a recent study (25) using a different model of inducing tissue inflammation in vivo lends credence to our findings. The ischemia/reperfusion-induced increase in E-selectin expression in the small intestine of wild-type mice was completely abolished in iNOS-/- animals. Given this situation, further studies are warranted to address the role of NO in adhesion molecule expression in vivo and in vitro to attempt to reconcile this controversy.

The CLP-induced E- and P-selectin expression was diminished in organs located within the peritoneum of iNOS-/- mice. This decreased expression of endothelial selectins was associated with a reduction in small intestinal MPO activity, indicating that selectins play an important role in PMN recruitment to the gut. In contrast, E-selectin expression on lung endothelium was reduced and P-selectin expression was unaffected in iNOS-/- mice (Fig. 4). However, MPO accumulation in the lungs of iNOS-/- mice was the same as in their wild-type counterparts. These findings suggest that either 1) P-selectin is sufficient for PMN accumulation in the lung or 2) leukocyte sequestration in the lung is independent of selectin expression. These results can be explained by organ-specific recruitment of leukocytes. Previous studies have indicated that blockade of selectin (or integrin) function during CLP results in reduced peritoneal accumulation of PMN, whereas lung PMN sequestration is unaffected (19, 30). Thus our data are consistent with the current consensus that in some organs (i.e., the small intestine) the classic adhesion pathway may predominate, whereas in the lung, PMN may adhere to pulmonary capillaries (capillary plugging) independently of adhesion molecule function (7).

In conclusion, the results of the present study indicate that the production of NO by iNOS plays a critical role in the expression of E- and P-selectin during polymicrobial sepsis in vivo. Specifically, the CLP-induced E- and P-selectin expression on endothelial cells is reduced in organs located within the abdominal cavity of iNOS-/- mice compared with their wild types. This reduction of endothelial selectin expression is associated with decreased PMN accumulation in the gut. Together, our findings indicate that NO plays an important role in modulating peritonitis-induced endothelial cell adhesion molecule expression and leukocyte sequestration in organ systems within the peritoneum.


    ACKNOWLEDGEMENTS

We thank Drs. D. N. Granger and P. Bauer for invaluable assistance in establishing the dual radiolabeled antibody technique in the laboratory. We thank R. Bateman for technical assistance with the NOx- chemiluminescence detection.


    FOOTNOTES

This work was supported by Grants MT-13940 and GR-12816 from the Canadian Institutes of Health Research (CIHR). C. Lush is supported by the Doctoral Research Award from the CIHR.

Address for reprint requests and other correspondence: P. R. Kvietys, Lawson Health Research Institute, Vascular Biology Program, 375 South St., Rm. C210, London, ON, Canada N6A 4G5 (E-mail: pkvietys{at}julian.uwo.ca).

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.

Received 22 March 2000; accepted in final form 25 August 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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