Annals of Burns and Fire Disasters - vol. XI - n. 1 - March 1998

ASSESSMENT OF CERTAIN NEUTROPHIL RECEPTORS, OPSONOPHAGOCYTOSIS AND SOLUBLE INTERCELLULAR ADHESION MOLECULE.1 (ICAM-1) FOLLOWING THERMAL INJURY

Faculty of Medicine, Mansoura University, Mansoura, Egypt

SUMMARY. Polymorphnuclear leukocytes (PMLs) play a key role in host defence, and phagocyte dysfunction has been associated with increased susceptibility to infections in patients with thermal injury. Intercellular adhesion molecule-1 (ICAM-1) plays a role in leukocyte accumulation and extravasation. Flow cytometric analysis (FCM) was used to study PML expression of IgG Fc-receptor III (Fey R111) as well as the complement receptors CRI (receptor for C3b) and CR3 (receptor for Obi) in 23 patients with large burns. Analysis of PML complement- and immunoglobulin-mediated phagocytosis of Candida albicans was performed in parallel using the phagocytic index. Plasma ICAM-1 was determined using ELISA. This study revealed a significant increase, with variable degrees, in CRI- and CR3-dependent fluorescence, complement-mediated phagocytosis of Candida albicans, and plasma ICAM- 1, starting on day 2 and continuing for about 20 days until normalization. In contrast Fcy RIII-dependent fluorescence and Ig-mediated phagocytosis were significantly decreased versus control values. These results demonstrate a significant change in PML opsonin receptor expression and opsonophagocytosis, documenting systemic activation of PMLs after large burns. In addition, elevation of plasma ICAM-1 may enhance the harmful effect of neutrophil activation due to leukocyte accumulation and extravasation following enclothelial damage in the skin and lung.

In spite of marked improvements in fluid resuscitation, respiratory care techniques, and other intensive care procedures introduced in the last decades, infection continues to be the leading cause of death in thermally injured patients.' Loss of the protective skin barrier, nutritional imbalance, and increased metabolic requirements contribute to the increased susceptibility to infection. In addition, thermal injury induces profound abnormalities in specific and unspecific immunity, exposing the patients to considerable risk of infection!
Polymorphnuclear leukocyte (PMLs) are effector cells essential for protection against bacterial and fungal infection? Immunoglobulins and complement factors serve as opsonins and facilitate phagocytosis via specific opsonin receptors. The most important opsonin receptors include Fcy RII and Fcy RIII for IgG, besides CRI and CR3 for the C3 split products C3b and ON, respectively! The expression of these receptors is modulated by chemoattractants and cytokines, as well as by injury and various diseases.
Human Fcy receptor III (FCRIII) or CD16-antigen is expressed on neutrophils, natural killer (NK) lymphocytes, and macrophages. Two genes are coding for this receptor, FcRIII-1 and FcRIII-2. The FcRIII-I mRNA encodes a protein with a short (four amino acids) cytoplasmic domain, whereas the FcRIII-2 mRNA encodes a protein with a cytoplasmic domain of 25 amino acids. The FcRIII-I protein is proteolytically split during post-translational processing and coupled to a phosphatidylinositol (PI) anchor, whereas the FcRIII-2 protein appears to be a transmembrane protein. Neutrophils appear to express only the Pllinked form of FcRIll, while NK lymphocytes and macrophages express only the transmembrane form of FcRIII.
The two distinct receptors for opsonic fragments of C3 on human phagocytic cells that have been identified designated CRI and CR3. CRI binds C3b with higher affinity than ON and has been found to be a membrane glycoprotein with an apparent m.w. of 205,000 to 250,000. This protein mediates the binding of Ob-coated particles and immune complexes to a variety of cells bearing the receptor, including neutrophils, monocytes, macrophages, B lymphocytes, a subset of T lymphocytes, and glomerular podocytes.' CR3 binds Obi-coated particles. CR3, a membrane heterodimer present on human PMLs, monocytes and null cells, consists of two non-covalently linked polypeptides with m.w. of 155,000 to 170,000 and 94,000." Leucocyte stimulation with a variety of agents augmented the expression of CRI and CR3.
Cell membrane expression of adhesion molecules is iniportant for cellular interactions, including interactions during an immune response." In addition, adhesion molecules can be detected in vivo," and soluble adhesion molecules may then inhibit binding between membrane-bound adhesion molecules and their ligands. 14 ICAM-1 is expressed on many different cells and its expression can be induced by IL-I and TNF-a.." Serum concentrations of ICAM-1 can be increased during immune or inflammatory disorders.
Alterations of PML chemotaxis," phagocytosis," oxidative metabolism," and intracellular killing" have been demonstrated after thermal injury.
The present study was performed with Egyptian burn patients in order to assess the time course of PM1- expression of the opsonin receptors Fcy RIII, CRI and CR3, opsonophagocytosis of PMLs, and plasma-soluble ICAM1 concentrations in the first 20 days post-burn.

Patients
This work was performed on twenty-three patients (12 males, 11 females) admitted to the burn Unit at Mansoura University Hospitals (age range, 14 to 75 yr; mean age, 27.1 yr; total body surface area [TBSA] burn, 15 to 85%; mean TBSA burn, 43.3%). All patients had been exposed to flames, eight patients also presenting inhalation injury. Nine patients died. The criteria of the patient population are shown in Table I.

All patients were treated with vigorous fluid resuscitation, careful attention to nutritional status, and ventilatory support when indicated. The care of burn wounds included the use of topical agents (povidone iodine and silver sulphadiazine) and early excision of deep burns, with subsequent grafting. Patients undergoing surgery received prophylactic antibiotic agents. Otherwise, antibiotics were used only to treat clinically evident sepsis. burn wounds were cultured three times a week and blood cultures were taken as indicated clinically. Sensitivity tests were performed and antibiotics were given intravenously. All patients received a tetanus toxoid booster on admission. Informed consent was obtained from all patients.
A control group was included comprising ten healthy laboratory workers (4 males, 6 females) of matched ages.

• Leukoeyte collection

Peripheral blood was drawn from the patients on post-burn days 2, 5, 10, 15 and 20. The PMLs were obtained following lysis of the erythrocytes using the method described by Duque et al., slightly modified. Briefly, 100 ~d of heparinized blood were mixed with 5 ml of lysing buffer (8.9 gj/1 NH4C', I g/l KHC03, and 3.72 g/l EDTA), and left at room temperature for 10 min. The leukocytes were then washed in phosphate buffered saline (PBS) containing 0.5% bovine serum alburnin (BSA, Sigma Chemical Co.). Leukocyte total and differential counts were obtained by a Coulter Counter Model Onyx (Coulter Electronics) and the leukocytes adjusted at 5 x 106 PMLs/ml.

For the labelling of FCy RIII (CD16), the DAKO antibody that reacts with 50-70 KDa glycoprotein expressed on granulocytes was used. CR3 (CD I I b) was stained using the DAKO-CD11b mouse antiliuman antibody; CRI (CD35) was stained using the DAKO-CD35 mouse antiliuman antibody (DAKO, Denmark).

PML surface antigens were labelled using monoclonal antibodies by the indirect immunotluorescence technique. Briefly, for each antigen investigated 100 [tl of 1:100 dilution of specific monoclonal antibody was added to 100 ~tl leukocytes and incubated on ice for 30 min. After washing twice with PBS, 100 ml of 1: 100 dilution of fluorescein isothiocyanate antibody (goat antimouse) conjugate (DAKO) was added and then incubated for 30 min on ice. After a final wash, the cells were resuspended in 0.5 ml PBS. A negative control was prepared in the same manner, omitting the receptor-specific monoclonal antibody.

The cells were analysed using an EPICS-PROFILE 11 (Coulter Electronics, Fl, USA). The laser excitation wave length was 488 nm and standard filter settings were used. Leukocyte subpopulations were differentiated by combined measurements of forward-angle and side-angle light scatter, and the monoclonal antibody specific fluorescence gated on PMLs to a separate histogram. The PML surface receptors were expressed as the mean fluorescence of the PML population after staining with receptor specific monoclonal antibodies minus the negative control prepared from the same blood sample.

• Candida albicans

1. Preparation
   C. albicans was fixed by ethanol 70% for 1 h, then suspended in PBS solution and adjusted to a count of 5 x 107 /MI.
2. Complement C3 opsonization

For the opsonization of C. albicans with complement, Na2-EDTA was added to pooled human serum (PHS) to bind divalent cations, before the PHS was observed twice with Na2-EDTA-washed C. albicans to remove antibodies against the fungi. The absorbed PHS was then reconstituted with Ca 2~ and Mg 2± by addition of 0.1 ml 100 mol/I CaC12, and 100 Mol/l MgC12 per ml PHS. The C. albicans (5 x 107/mi) was then rotated at 37 'C for 45 min with PHS. The C3-opsonized C. albicans was washed twice, counted, and resuspended in PBS to a final concentration of 5 x 108 fungi/ml.

3. Immunoglobulin opsonization of C. albicans

The PHS was heated at 56 'C for 30 min to inactivate the complement. The C. albicans (5 x 10'/ml) was then rotated at 37 'C for 45 min with heated PBS, and the fungi were washed, counted and adjusted in PBS to 5 x 101/ml.

4. Phagocytosis

One hundred microlitres of PML suspensions were mixed with 100 ml of preopsonized C. albicans, before Hank's balanced salt solution (HBSS) containing 0.5% bovine serum alburnin (BSA) was added to a final volume of I ml. This provided an initial fungus to PML ratio of 10:1. The mixtures were rotated at 37 'C for 15 min. The neutrophilic suspension was then spread and stained by Leishman stain. Phagocytosis was measured cytomorphologically by determining the C. albicans phagocytic index according to Ballart et aL,` using the following equation:

• ICAM-1 assay

Peripheral blood samples were collected on EDTA, and centrifuged at 1000 g for 15 min within 2 h. Plasma was stored at -70 'C until analysis. Plasma concentrations were determined (in duplicate) by commercially available ELISA Kits (Biosource Europe S.A, Belgium). The minimum detectable concentration was estimated to be 0.3 ng/ml.

The data in this study were processed and analysed by SPSS PC version 6 under Windows. Central value and dispersion were represented by mean ± SEM. Analysis of difference is any two categories was performed using the Mann-Whitney-U test.

PML- expression of Fey RIII

The patient PM1- expression of Fey RIII was decreased to 65.7% of control values at admission and remained low for the first 20 days (Fig. M, Table II). PM1- expression of CRs
The patient PM1- expression of CRI increased to 162.9% of control values at 2 days, and then gradually decreased to control levels at 20 days (Fig. ]B, Table II).

The expression of PM1- CR3 increased by 199% on day 2 and remained high during the first 20 days (Fig. X, Table II). Both control and patient PN1L CRI-dependent and CR3-dependent fluorescence were shown to be monophasic throughout the investigation period.

Phagocytosis

The PML Ig-mediated phagocytosis of C. albicans decreased to 84.7% of control values on day 2. The lowest Ig-mediated phagocytosis was observed on day 10, with a reduction of 28.1% compared with controls (Fig. 2A, Table III).
The patient PML complement-mediated phagocytosis of C. albicans was increased by about 54% of control level and remained high for the first 20 days (Fig. 213, Table III).
The patient PML phagocytosis of C. albicans opsonized with PHS (i.e. in the presence of both IgG and complement) increased by about 3 1 % of control values and remained higher than that of control for 20 days (Fig. 2C, Table III).

Plasma levels of ICAM-1
Plasma ICAM-1 increased by 31% of control values on day 2 and remained high for the first 20 days (maximum level on day 10 (Fig. 3, Table IV).

Neutrophil activation by chemoattractants, enzymes and bacteriologically-derived peptides release Pl-linked FeRIll into plasma and in tissues with active inflammation.'PI-Iinked FcRIll mediates exocytosis of neutrophil granule proteins but does not mediate the initiation of the respiratory burst.` The present study demonstrates that expression of PML Fcy RIII following burn injury decreased on day 2 and remained low for the first 20 days. This result is thus indicative of a systemic activation of these cells, which induce shedding of this receptor, a finding in agreement with Vindenes and Bjerknes.

A marked and sustained increase in the expression of the complement receptors CRI and CR3 was documented in the present study, The increase in fluorescence that depends on complement receptors was always observed as a monophasic peak, indicating that all the cells had been activated by a stimulus that was systemic. Moore et al." and Vindenes and BjerkneS26 reported similar results. The increased expression of CR3 is an important part of neutrophil priming and activation as this molecule is critically involved in adherence of PML to the endothelium. This is a necessary initial step in the emigration of PMLs from the circulation into the tissues. In addition, both CRI and CR3 are necessary for optimal phagoeytosis of complement-coated bacteria and immune complexes.

Previous studies have shown that a variety of mediators such as the complement split product C5a '27 TNF-(1,28 IL-8,' granulocyte macrophage colony-stimulating factors' and endotoxin," which might be locally produced at sites of infection, are all capable of increasing complement receptor expression.

There are intracellular pools for both CRI and CR3, but the intracellular locations for these pools are distinct. The pool for C3 co-sediments with specific granules, while the pool for CRI does not."The increased receptor expression occurs within minutes and represents a translocation of presynthesized receptors from intracellular pools to the surface rather than a new synthesis. Consequently, the increased expression of PML CRs following thermal injury strongly suggests PML degranulation. This is consistent with earlier reports by Alexander" and Davis et al.
This study revealed that plasma ICAM-1 levels were elevated during the first 20 days post-burn, with a maximum level on day 10. This finding could be related to the activation of the inflammatory cytokines such as IL-16, TNF-P and INF-a, which induce or enhance the expression of both membrane and soluble ICAM-l."," Jaeschke et al." reported an increased plasma ICAM-1 level in endotoxin-challenged mice. In acute and chronic inflammatory processes, fibrin deposition and leukocyte accumulation are classic histopathological hallmarks. Fibrin deposition on vascular endothelial cells (EQ can result in the upregulation of EC ICAM-1, which is an important ligand/receptor for CDllb/CDl8 expressed on neutrophils. Fibrin stimulation of EC increased their adhesiveness for PMLs.` Circulating levels of soluble endothelial cell adhesion molecules may reflect the magnitude of expression of their membrane-bound counterparts." Mulligan et al." have emphasized the role of ICAM-1 in the events that lead to neutrophil-mediated vascular injury of dermis and lung after thermal trauma to the skin. These would cause cessation of neutrophil movement along the endothelial cells and transmigration.
Systemic activation of PMLs may have harmful effects on the host. Increased PM1- CR3 expression as well as ICAM-1 plasma level may cause increased neutrophil adhesiveness and the formation of leukoeyte microemboIi that concentrate in the first capillary bed encountered, the lung There is also depression of neutrophil chemotaxis.` Activated PMLs are known to degranulate and release lysosomal enzymes as Well as oxygen radicals toxic to surrounding tissues. Injury~related PNNI activation can sustain and be further strengthened by subsequent superimposition of infectious foci (e.g. burn wound sepsis" or by transintestinal transport of LPS or micro-organisms .
This recruitment and activation of PMLs in the course of thermal injury may exceed physiological needs and induce PN1L tissue infiltration and destruction. We therefore recommend that the apparent source of neutrophil-activating substances, the burn wound, should be excised as early as clinically feasible in order to free the patient of the burden of systemically activated neutrophils` and early initiation of enteral feeding. The use of anti-proinflammatory cytokines and/or anti-ICAM-1 needs further investigation.

RESUME. Les leucocytes polymorphnucléaires (LPMs) jouent un rôle fondamental dans la défense de l'hôte, et le dysfonctionnement phagocytaire a été associé à la susceptibilité augmentée à l'infection dans les patients brûlés. La molécule-1 d'adhésion intercellulaire joue un rôle dans l'accumulation leucocytaire et l'extravasation. L'analyse cytométrique du flux a été utilisée pour étudier l'expression par les LPMs du récepteur Fe 111 IgG (Fcy RIII) comme aussi des récepteurs du complément CRI (récepteur pour C3b) et CR3 (récepteur pour C3bi) dans 23 grands brûlés. Cette étude a indiqué une augmentation significative, avec une certaine variation, de la fluorescence, de la phagocytose obtenue avec le complément de Candida albicans, et du plasma ICAM, après le deuxième jour et pour les vingt jours suivants. La fluorescence dépendante de la FCy RIII et la phagocytose obtenue avec l'Ig démontraient, au contraire, une augmentation significative par rapport aux valeurs témoins. Ces résultats indiquent une variation significative dans l'expression du récepteur de l'opsonine des LPMs et dans la phagocytose, ce qui confirme l'activation systémique des LPMs après les grandes brûlures. En outre, l'élévation de lICAM-1 plasmatique peut augmenter l'effet nuisible de l'activation des neutrophiles.

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