Decreased neutrophil l-selectin expression in patients with systemic inflammatory response syndrome

Najma A. Ahmed, MD, PhD
Nicolas V. Christou, MD, PhD

Clin Invest Med 1996; 19 (6): 427-34.

[résumé]

Dr. Ahmed is a resident in general surgery and Dr. Christou is a professor in the Department of Surgery, McGill University, Montreal, Que.

This article was presented at the 1995 Annual Meeting of the Canadian Society for Clinical Investigation and of the Royal College of Physicians and Surgeons of Canada, Montreal, Que., Montreal, Sept. 13 to 17, 1995.

(Original manuscript submitted Jan. 1, 1996; received in revised form May 22, 1996; accepted June 3, 1996)

Paper reprints may be obtained from: Dr. Nicolas Christou, Professor of surgery, Room C5.53, Royal Victoria Hospital, 687 Pine Ave. W, Montreal QC H3A 1A1; fax 514 843-1430; nchristou@is.rvh.mcgill.ca

Contents

Abstract

 Objective: To determine whether the expression of l-selectin is reduced in patients with systemic inflammatory response syndrome (SIRS) compared with control subjects, to test the effect of exudation of neutrophil l-selectin expression, and to measure soluble serum concentrations of shed l-selectin in the two groups.

Design: Prospective study.

Setting: Intensive care unit in a tertiary care hospital (Royal Victoria Hospital, Montreal).

Patients: Twenty-five patients with SIRS and 20 healthy, age-matched controls.

Interventions: Collection of exudate neutrophils from skin window and circulating neutrophils from venous blood.

Outcome measures: Neutrophil l-selectin levels, measured with the use of fluorocytometry, and soluble l-selectin levels, measured with the use of enzyme-linked immunosorbent assay.

Results: There is a significant reduction in l-selectin expression on circulating neutrophils in patients with SIRS, compared with control subjects. Exudation of neutrophils to an extravascular site resulted in a dramatic down-regulation of l-selectin in both groups. Serum levels of soluble l-selectin were higher in patients with SIRS than in control subjects.

Conclusions: The loss of l-selectin may be partly responsible for reduced neutrophil exudation to extravascular sites in patients with SIRS.

Résumé

 Objectif : Déterminer si l'expression de la l-sélectine est diminuée chez les patients atteints du syndrome de réponse inflammatoire systématique (SRIS) en comparaison avec un groupe témoin; étudier l'effet de l'exsudation des neutrophiles sur l'expression de la l-sélectine des neutrophiles; et comparer la concentration sérique de l-sélectine entre les deux groupes.

Devis : Étude prospective.

Cadre : Unité de soins intensifs dans un hôpital de soins tertiaires (Royal Victoria Hospital, Montréal).

Sujets : Vingt-cinq sujets avec SRIS et 20 sujets normaux appariés pour l'âge.

Intervention : Prélèvements de sang veineux (des neutrophiles circulants) et de liquide au niveau d'une fenêtre cutanée (des neutrophiles exsudatifs).

Variables mesurées : Mesure par fluorocytométrie des niveaux de l-sélectine dans les neutrophiles et mesure par ELISA des niveaux de l-sélectine soluble.

Résultats : En comparaison avec les témoins, une diminution significative de l'expression de la l-sélectine a été notée dans les neutrophiles circulants des sujets avec SRIS. Dans les deux groupes, l'exsudation des neutrophiles vers un site extra-vasculaire était associée à une régulation négative très marquée de la l-sélectine. Les niveaux sérique de l-sélectine soluble étaient plus élevés dans les SRIS que chez les témoins.

Conclusion : La perte de l-sélectine pourrait être en partie responsable pour la diminution de l'exsudation des neutrophiles vers les sites extra-vasculaires chez les sujets avec SRIS.

[Table of contents]

Introduction

 Systemic inflammatory response syndrome (SIRS) is common in the modern intensive care unit (ICU), affecting 15% to 30% of all patients admitted to the ICU. The pathogenesis of SIRS is related, in part, to the elaboration of cytokines, to aberrant immune-cell function and to inadequate oxygen extraction and metabolism. These factors interact in a complex manner and culminate in a severe dysregulation of immune and metabolic response of the host. The clinical expression of this dysregulation results in SIRS.[1] Persistent or overwhelming SIRS leads to progressive multiple organ dysfunction syndrome (MODS), which accounts for approximately 70% of deaths in the ICU.[2]

Neutrophils are important effector cells in the host response to infection or injury. They are efficient phagocytes and possess a potent arsenal of cytokines, which act as paracrine, autocrine and endocrine regulators of immune and other cells. Immune dysregulation associated with neutrophil dysfunction has been implicated in the pathogenesis of SIRS and MODS.[3­9] Site-specific neutrophil activation and migration are tightly regulated processes that are essential to eradication of pathogens and recovery of the host. The migration of neutrophils is mediated in large part by the complementary interaction of cell-surface molecules expressed on neutrophils and endothelial cells, known collectively as cell adhesion molecules (CAM). CAM-mediated interactions, coupled with chemotactic signals released by lysed bacteria and secreted by other phagocytes and endothelial cells, direct the site-specific engagement of neutrophils with endothelial cells and the subsequent diapedesis into the parenchymal cell compartment.[10­17]

Neutrophil adhesion molecules comprise two major classes: the selectins and integrins. l-selectin is a lectin glycoprotein expressed on almost all leukocytes, except a class of memory B-lymphocytes. It is required for efficient homing of lymphocytes to lymph nodes and other immune organs. Neutrophil l-selectin mediates the initial contact and rolling of neutrophils within endothelial-lined vessels under conditions of flow.[18­22] This initial l-selectin-mediated contact is essential to subsequent neutrophil extravasation to the parenchymal cell compartment.[23] The transmigration of neutrophils has been shown to be greatly inhibited in l-selectin-deficient mice.[24] Furthermore, l-selectin is being seen as an important receptor in signalling intracellular activation. This contention is supported by experiments demonstrating that cross-linking of l-selectin results in greater superoxide anion generation, increased calcium ion flux and up-regulation of beta-integrins.[25­30]

In this study, we tested the hypothesis that the expression of l-selectin on circulating neutrophils is decreased in patients with SIRS, compared with the expression in patients without the syndrome. We also compared the expression of neutrophil l-selectin on intravascular and exudative neutrophils and measured soluble levels of shed l-selectin in patients with SIRS and control subjects.

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Materials and methods

Subjects

Twenty-five patients who met the clinical criteria for SIRS admitted to the ICU at the Royal Victoria Hospital were compared with 20 healthy, age-matched control subjects recruited from the community. We used the criteria for SIRS of the American College of Chest Physicians and the Society of Critical Care Medicine: a temperature of less than 36.5°C or more than 38.5°C, tachypnea of greater than 20 breaths per minute or ventilator dependence, tachycardia of greater than 90 beats per minute, leukocyte count of more than 11 000 cells/dL (11.0 × 109/L) and evidence of organ dysfunction.[31] All patients were studied within 24 to 48 hours of meeting the clinical criteria for SIRS. Patients were excluded if they had undergone chemotherapy or radiotherapy in the previous 3 months, had a history of long-term steroid use or were HIV-positive. Acute physiology and chronic health evaluation scores (APACHE II) were collected for each patient on the first day of study. Control subjects were excluded if they had consumed drugs or alcohol in the previous 48 hours, had a severe chronic illness requiring use of immunosuppressive drugs or steroids, or had known cancer. The study was approved by the Committee of Human Experimentation of the Royal Victoria Hospital, Montreal, and informed consent was obtained in all cases.

Collection of skin window fluid

Skin window chambers were applied to the volar aspect of the forearm, as described elsewhere.[32] Briefly, the forearm was sterilized with tincture of iodine and then cleaned with a 70% isopropyl alcohol solution. A vacuum suction of 360 mm Hg was applied through a plastic template for 30 to 60 minutes until four even blisters measuring 1.0 × 1.0 cm had formed. These blisters were unroofed with sterile scissors, and a template consisting of four 1.0 × 1.0 open-bottomed chambers was tightly applied with wide adhesive tape. The chambers were filled with 10% autologous serum, and the superior apertures were sealed. The exudate fluid was aspirated 14 to 16 hours later. The chambers were rinsed three times with normal saline solution, and the fluid was transported immediately on ice to the laboratory. The neutrophils were sedimented at 3000 rpm at 4°C for 5 minutes, and the supernatant fluid was frozen immediately at -70°C in a Revco freezer for batch analysis of soluble l-selectin levels.

Neutrophil l-selectin expression

Venous blood was drawn into tubes coated with ethylenediamine tetra-acetate (EDTA) and was kept at 4°C until processing, which was always conducted within 1 hour of obtaining the specimen. Cellular viability of exudative neutrophils obtained from skin window chambers with the use of propidium iodide exclusion[33] (Sigma, St. Louis MO) was determined to be more than 95%. Expression of surface l-selectin on circulating and exudative neutrophils was determined by fluorocytometry. Specific murine antihuman monoclonal or isotype control antibodies were incubated in the presence of whole blood neutrophils. l-selectin expression was determined with the use of two different monoclonal antibodies that are specific to different l-selectin epitopes (Leu-8 PE*, Becton Dickinson, Mountainview, Calif., and TQ1-RDI, Coulter Immunology, Miami). IgG2a-PE* (Becton Dickinson, Mountainview, Calif.) was used as isotype control. Neutrophils were centrifuge-sedimented, washed with phosphate-buffered saline solution and fixed in paraformaldehyde for at least 1 hour at 4°C. Cellular excitation for fluorocytometry was performed with the 488-nmol/L line of a 15-mW argon ion laser. Band filters of 585 nmol/L were used. Forward versus side scattergrams were used to gate leukocyte populations electronically. Fluorescence intensity was determined on a log scale from 1 to 10 000, on the basis of 10 000 events acquired at a rate of 60 mL per minute. Signal processing and data analysis were completed with Consort 30 software (Becton Dickinson, Mountainview, Calif.). Mean channel fluorescence and the percentage of antibody-positive cells were determined by subtracting isotypic control fluorescence values.

Serum soluble l-selectin levels

Whole blood samples were collected by venipuncture and centrifuged immediately at 3000 rpm for 20 minutes. The supernatant, consisting of serum, was immediately frozen at -70°C until batch analysis for soluble l-selectin. Exudate fluid from skin window chambers was collected as described above and frozen immediately at -70°C. An enzyme-linked immunosorbent assay (ELISA) (Bender Medsystems, Vienna, Austria) was used for these determinations.34 Serum and skin window fluid samples were incubated on microtitre plates adsorbed with murine monoclonal antibody to human soluble l-selectin. Murine monoclonal antisoluble l-selectin conjugated with horseradish perioxidase was used to detect soluble l-selectin. After incubation, the unbound enzyme-conjugated anti-l-selectin was removed by washing. The colouring reaction was terminated by adding 4N sulfuric acid. Absorbance was determined spectrophotometrically with a Titeterk, Multiscan ELISA plate reader (Flow Laboratories, Mississauga, Ont.) at 450 nm. A standard curve of concentration of known standards against absorbance was plotted. The level of soluble l-selectin in each plasma and exudate fluid sample was calculated from the standard curve after correction for dilution.

Statistical analysis

Differences in continuous variables between groups were tested with the use of analysis of variance with Schiefe's test for multiple comparisons. Spearman rank correlations (rs) were used to determine the relation between soluble serum l-selectin level and the expression of l-selectin on neutrophils.

[Table of contents]

Results

The demographic and clinical data for the patient cohort are described in Table 1.

We found significantly lower expression of l-selectin on circulating neutrophils in patients than in control subjects. This trend was seen for both Leu-8 (p = 0.001) and TQ-1 (p = 0.00001, Fig. 1 and Fig. 2). Conversely, serum soluble levels of l-selectin were significantly higher in patients than in control subjects (p = 0.00001, Fig. 3). There was an inverse correlation between circulating neutrophil expression of TQ-1 and serum soluble l-selectin levels (rs = 0.60, p = 0.02, Fig. 4). There was a similar, but weaker, inverse correlation between circulating neutrophil Leu-8 expression and serum soluble l-selectin levels (rs = -0.27, not significant, Fig. 5). Exudation of neutrophils from the intravascular compartment to skin window chambers caused a loss of l-selectin expression in both patients and control subjects. There was no significant difference between exudative neutrophil l-selectin expression between patients and control subjects (Table 2). Soluble levels of l-selectin were 15 to 40 times lower in skin window fluid than in serum. Concentrations of soluble l-selectin in skin window fluid were similar in patients and control subjects (Table 3). There was no significant correlation between APACHE II scores and l-selectin expression on circulating or exudative neutrophils in either patients or controls.

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Discussion

Our finding of significantly lower expression of l-selectin on circulating neutrophils in patients with SIRS than in control subjects is consistent with the intravascular activation of neutrophils that is known to occur in critically ill patients.[35,36] Similarly, l-selectin expression on umbilical cord blood granulocytes and monocytes is decreased in newborn infants with acute bacterial infection, compared with control newborns.[37] In contrast, premature infants, who are at greater risk of infectious complications than term infants, have higher, rather than lower, neutrophil l-selectin expression than term newborns.[38]

Tumour necrosis factor-* and other cytokines cause shedding of neutrophil l-selectin in vitro.[39] Elevated plasma levels of various cytokines, and in particular tumour necrosis factor-*, have been documented in SIRS.[40] Cytokines, complement products and bacterial cell-wall fragments act in concert to cause intravascular neutrophil activation and shedding of l-selectin. In this study, neutrophil l-selectin expression did not correlate with disease activity, as measured by APACHE II score. Intravascular neutrophil activation also occurs in systemic lupus erythematosus; however, in keeping with our results, there is no correlation between disease activity and l-selectin expression.[41]

We recently showed that neutrophil extravasation to skin window chambers was reduced by 70% in patients with SIRS.[42] The reduction in neutrophil l-selectin documented here may partially account for this finding. Intravascular shedding of l-selectin decreases the efficiency of margination, preventing rolling of neutrophils on endothelial cells. In vitro studies show that neutrophil rolling depends on endothelial expression of e-selectin, which engages sialyated carbohydrate ligands on the neutrophil surface.[43] Furthermore, l-selectin-mediated rolling is required for subsequent beta-integrin-mediated firm adhesion and transmigration.[23] The importance of l-selectin to neutrophil transmigration is reinforced by the dramatic reduction in neutrophil exudation to inflammatory sites in l-selectin-deficient mice.[24] We hypothesize that, in the current study, loss of l-selectin on circulating neutrophils lessens their ability to localize to sites of inflammation by reducing rolling on inflamed endothelium. l-selectin-mediated contact is only one among many interacting steps that finally result in neutrophil extravasation. Once firmly attached, neutrophils must orient along chemotactic and haptotactic gradients, secrete proteolytic enzymes capable of digesting extracellular matrix, transform into motile cells and migrate through the vessel wall.[44] This complex interplay, now the subject of intensive research, is orchestrated by cytokine and other mediators, chemotactic signals and the site-specific expression and engagement of CAMs.

Levels of shed serum l-selectin were significantly greater in patients than in control subjects, a finding consistent with previous reports in patients with SIRS and with AIDS.[45,46] Elevated soluble l-selectin levels may be a marker for leukocyte activation, and their clinical relevance and applicability are being studied.[46] The precise physiologic relevance of circulating adhesion molecules remains to be determined. Whether they are active or inactive fragments, whether these soluble fragments are available to bind counter-receptor-bearing cells and what mechanisms clear these fragments are the subjects of current investigation.

Levels of soluble l-selectin were much lower in exudative skin window fluid than in plasma, suggesting that shedding of l-selectin from the neutrophil surface occurs in the peripheral circulation, as a prelude to diapedesis. This view is consistent with the current paradigm of neutrophil extravasation. Extravasation of neutrophils to skin window chambers resulted in a dramatic reduction in neutrophil l-selectin, to almost unmeasurable levels. Similar results have been reported in mice and normal human subjects.[47­49] Ours is the first report to document expression of l-selectin on exudative neutrophils in this specific patient population.

We find it curious that correlations between the two epitopes of l-selectin and shed l-selectin were so dissimilar. The correlation with the TQ-1 epitope was twice as great as that with the Leu-8 epitope (Figs. 4 and 5). This suggests that simple cleavage of the extracellular domain of l-selectin is not the only or entire mechanism accounting for the down-regulation of l-selectin, as has been previously hypothesized.[50­52] Rather, decreased neutrophil l-selectin may be partially accounted for by conformational changes or internalization of part of the molecule, whereas other parts may be cleaved and circulate as soluble l-selectin. Recent studies of human neutrophils and eosinophils suggest that there are different functional epitopes of l-selectin in these two cell populations.[53] These observations may also apply to functionally different classes of neutrophils or functionally different epitopes of l-selectin on a homogenous population of neutrophils.

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Conclusions

l-selectin expression was significantly lower in patients with SIRS than in control subjects, whereas soluble shed l-selectin levels were higher in patients with SIRS than in controls. Although neutrophil l-selectin did not correlate with APACHE II scores, TQ-1 expression of l-selectin correlated inversely with soluble shed l-selectin levels. The diminished exudation of neutrophils we previously documented in patients with SIRS may be related to decreased l-selectin expression and, as a consequence, decreased margination and rolling.

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