Ann. Medit. Burns Club - vol. VII - n. 3 - September 1994

ALTERED NEUTROPHIL FUNCTION IN PATIENTS WITH MAJOR BURN TRAUMA

Pralat U.*, Lips U.*, Pallua N.**, Stangel W.'

Department Anaesthesiology* Clinic of Plastic and Reconstructive Surgery**
Blood ban k- I mm un e haematology***
Hannover Medical School, Hannover, Germany

SUMMARY. After severe burn injury, large amounts of tissue debris, endotoxins and micro-organisms have to be eliminated. Growing exhaustion of the non-specific immune system leads to the secondary immunodeficiency syndrome with polymorphonuclear leucocytes (PMN) profoundly suppressed. Twenty-seven burn patients were studied with a total of 221 measurements. The results showed that chemotaxis in the surviving patients was significantly higher than in the non-surviving group. Superoxideanion and hydrogen peroxide play a suspected role in the pathogenesis of the adult respiratory distress syndrome (ARDS). However, hydrogen peroxide production after stimulation with formyl-methionyl-leucyl-phenylalanin (FMLP) was nearly three times higher in non-surviving patients than in survivors. Patients with a regular clinical course show lower superoxideanion generation than non-survivors after stimulation with FMLP. These results indicate that non-surviving patients displayed reduced chernotaxis, while the generation of possibly damaging oxygen radicals was significantly enhanced in comparison with patients with a regular clinical course.

Acute thermal injury produces a variety of adverse effects in humans. Paramount among these effects is a dramatic depression in host resistance such that the burn patient is susceptible to infection by a number of opportunistic pathogens. Intensive studies over the past several years have shown that this increased incidence of infection is due to defects in non-specific defence functions, such as neutrophil and macrophage phagoeytosis (20, 22) and bacterial activity (2), as well as to an inhibition of specific immune functions, such as cell-mediated immunity (17, 20, 3 1) and antibody formation (2, 13, 17). Available evidence shows that although specific immune functions play an equally important role in the patient's defence network, the nonspecific neutrophil and macrophage activities are immensely important in controlling infection in the burn patient.
Sepsis and the adult respiratory distress syndrome (ARDS) continue to be major life-threatening complicalions in patients with extensive thermal injury (1, 14). The decrease in host resistance to infection is obviously related to a depression of both humoral and cellular components of the host defence system. Although most parts of the immune system that have been studied after thermal injury show impairment, cell-mediated immunity turned out to he most consistently and profoundly suppressed (4, 8). Polymorphonuelear leucocytes (PMN) form a major part of the non-specific first line of defence. PMN are a potential source of oxygen free radicals, which are produced in connection with the respiratory burst of these pha~ gocytic cells (Fig. 1). Intracellular killing by PMN requires the uptake of oxygen by the phagocytic cell. The cytoplasm of the leucocytes is protected from oxygen free radical damage by the presence of the so-called radical scavengers, superoxide dismutase and catalase. Through a series of enzyme reactions including NADPH oxidase, superoxide dismutase and catalase, the oxygen is progressively reduced to superoxide, hydrogen peroxide, hydroxyl ions and singlet oxygen. If, however, oxygen free radicals leak into the interstitial space where the concentration of scavengers is very low, damage to cell membranes is very likely to occur. Chemotaxis is presumed to be an important function of PMN in protection against invasive agents. Even leucocytes fully capable of ingesting and killing micro-organisms, tissue debris and endotoxins can be incapacitated if unable to sense and be directed toward the antigen. In order to characterize secondary immunodeficiency following severe thermal injury, we investigated PMN ability as regards chemotaxis and the generation of supetoxideanion and hydrogen peroxide iii surviving and nonsurviving patients.

Patients: in all, 27 patients suffering from second- and third-degree burns in more than 35% BSA (maximum 92%) were studied. There were 19 male and 8 female patients ranging in age from 19 to 68 years (mean age 39.7 ± 9.7 yr). A total of 149 measurements were analysed from the group of 16 surviving patients, and 72 samples were taken from the 11 non-surviving patients, who eventually died because of sepsis and ARDS. In this group there was an APACHE 111 (11) score of 76.7 ± 22.8. Nine patients also suffered from inhalation trauma. The surviving patients had an APACHE Ill (11) score of 40.2 ± 12.8. No patient developed sepsis and only three were observed to have inhalation trauma.
Informed consent was obtained from all patients or their relatives.

Samples were taken daily on 11 consecutive days. We investigated PMN chemotaxis against formyl-methionylleucyl-phenylalanin (FMLP) and yeast-activated serum (YAS) according to Chenowth (3) in "under agarose technique". The ability of PMN ability to generate H202 and 02 - was tested after stimulation with opsonized zymosan, FMLP and zymosan-activated serum (16, 27). Evaluation Of 02- generation was performed by observing cytochrome C reduction. Assessment of H202 production was carried out kinetically, using horseradish peroxidase mediated reduction of scopoletin. Statistical analyses were performed utilizing Student's t-test after establishing medians in each series.

A total of 27 patients were studied, involving a total of 221 measurements. Matching normal volunteers were used as controls. Results are shown in Figs. 2-4. Chemotaxis in response to FMLP in cells from surviving patients was 1.02 ± 0.27 mm and significantly higher than in PMN from the non-surviving patients, where it reached 0.67 ± 0.22 mm (Fig.2). There was no significant difference between the two groups using YAS (surviving group cells, 0.62 ± 0.20 min; non-surviving group cells, 0.59 + 0.24 min, p<0.001). Fig. 3 shows 11,0, production after stimulation with FMLP and opsonized zymosan. In PMN from the non-surviving patients there was a nearly threefold higher H202 generation (0.89 ± 0.19 nmol/106) than in PMN from surviving patients (0.3 ± 0.09 nmol/106) wh 1 ile using FMLR There was no difference between the two groups after stimulation with opsonized zymosan.
Fig. 4 indicates a significant difference between superoxideanion generation between the two groups of patients. Surviving patients showed lower 02- production (0.68 ± 0.19 mnol/106) than non-surviving septic patients (1.43 ± 0.43 nmol/106) while stimulated with FMLP. After YAS/ZAS stimulation we find in survivors less 0, - generation (0.31 ± 0.07 nmol/106) than in non-survivors (1.88 ± 0.24 nmol/106).

Severe burns are always followed by infections which may lead to sepsis, ARDS and multi-organ failure. The complications are considered to be closely related to the post-burn immunodeficiency syndrome (14). Increased suspectibility to infections in burn patients occurs in severe conditions, namely the open contaminated wound, increased metabolic requirements, decreased nutritional intake, loss of plasma protein and suppression of the patients' defence mechanisms (8, 15). However, defects do not occur in isolation. As a result it is often difficult to determine whether a particular abnormality is the primary event, an impairment which is the consequence of another host defence abnormality, or the result of infections, metabolic derangements or other secondary effects of the burn trauma.
Based on the results of immunological research, activated leucocytes are now regarded not only as functioning in specific and nonspecific immune defences, but also as having a great potential for causing deleterious effects on the host organs.
Neutrophils (24) and macrophages are thought to play especially important key roles in the pathogenesis of acute lung failure after sepsis and trauma. By an excessive liberation of lysosomal enzymes, vasoactive eicosanoids and toxic oxygen derived radicals they can cause increases in permeability, changes in vascular tonus, and damage to the endothelium. of the lung vessels (6, 9, 10, 19, 29, 30).

As shown by many investigations, the sequestration of PMN in the lung is a crucial prerequisite for the full expression of the permeability lesion in ARDS. During lung failure one can regularly find an excess of adherent PMN in the pulmonary microvasculature, lung lymph and bronchoalveolar lavage. It has been demonstrated that, in the bronchoalveolar lavage of patients with ARDS, neutrophils constitute nearly 70% of the recovered cells in comparison to 14% in normal volunteers and ventilated control patients (28). The percentage of neutrophils also correlated with A,D02 and lung protein permeability.
The role of polymorphonuclear leucocytes in host resistance to infection is receiving much attention at the present time. Immediately after antigen stimulation, i.e. by bacteria and injured tissue, PMN adhere to blood vessel walls and then emigrate into the tissues. This movement, chemotaxis, is unidirectional, with no return of the cell to the circulation, and it is facilitated to a large degree by complement. Following phagocytosis, the intracellular destruction of phagocytized bacteria by PMN is realized by a "respiratory burst" of cellular activity. However, the ability of PMN to generate superoxideanion and hydrogen peroxide is extremely interesting as regards respiratory failure (14).
Our findings in severely burned patients indicate the quantity and quality of parts of granulocyte impairments. In severely compromised burn patients with fatal outcome, FMLP mediated superoxideanion production was augmented and correlated with the burned body surface area and septicaernia (Fig. 4). A similar increase in H202 generation ican also be observed in this group (Fig. 3).
The reason for this may be a possible defect in detoxification of oxygen radicals with its suspected role in the pathogenesis of ARDS. After activation of neutrophils, the cells undergo a "respiratory burst" with an associated increase in oxygen consumption. In conjuction with this, neutrophils have shown to secrete both superoxideanion and hydrogen peroxide. Obviously, patients suffering from sepsis following burns show alteration in oxygen metabolisms at an early stage. Granulocyte stimulation with different agents proved that the amount of superoxideanion production depends on the kind of stimulus (5). Tschaikowsky and Ruegheimer (25) have shown that in septic patients there is an increase in FMLP receptors. It is thought that the integration of pre-formed receptors following activation of PMN by intrinsic or extrinsic factors or micro-organisms might be responsible.
Our patients with fatal outcome display compromised chemotaxis to FMLP, while the generation of possible damaging oxygen radicals is significantly enhanced in comparison to patients with a regular clinical course. Defects in neutrophil bactericidal capacity were reported by Alexander in 1971 (2) to be evident in burn patients during periods of septicaemia. We found that reduced chemotaxis confirms the study of Warden et al. (26), who described leucocyte chernotaxis as being inversely correlaled with burn size during the first 72 hours post-burn. After 72 hours, leucocyte chernotaxis directly correlates with clinical status and is highly predictive for mortality (26). In view of the small number of patients that we studied, we are not able to confinn this, but since mortality in our patients was largely due to infection, these findings suggest that suppression of leukocyte chernotaxis may explain the susceptibility to opportunistic infection in thermally injured patients. Grogan (7) monitored neutrophil ingestion and bactericidal capacity in 35 burn patients, using Pseudomonas aeruginosa as the test micro-organism, and found that defects were common in the phagocytes of patients with burns in more than 30% BSA. However, for the first five days, there was no acute alteration. After this initial period, phagocytosis and killing acti-vity were significantly reduced. Nevertheless, 77% of the septic patients exhibited a reduced neutrophil bactericidal capacity (7). In general, killing effects were detected more often than an effect in the ingestive phase of phagocytosis, although the latter was discovered in a statistically significant number of tests (7).

In agreement with Alexander et al. (1) and Solomkin et al. (23) our study shows a decrease in neutrophil bactericidal capacity which was higher in non-surviving patients. These patients died because of respiratory failure and sepsis following heavy wound colonization.
Other investigators have reported normal neutrophil function in patients who were severely ill with septicaemia. Messner et al. (12) and Solberg (22) studied non-burned patients with infections whose transient neutrophil bactericidal defects returned to normal when the patients had recovered from the infection.
However, the pathophysiology of decreased chemotaxis and increased metabolic response remains to be elucidated and a casual relationship between our findings and subsequent infection can only be theorized. We believe that monitoring chernotaxis and superoxide and hydrogen peroxide provides an indirect measure of part of the basic membrane and cytoskeletal functions upon which stimulated migratory functions are based. Speculating further, one miight expect that therapy to control stimulated secretory responses of the circulating PMN could effectively limit the generalized cellular dysfunction that accompanies thermal injury with its excessive complement activation and release of cytokines.

RESUME. A la suite des brûlures sévères, il faut éliminer de grandes quantités de débris tissulaires, d'endotoxines et de micro-organismes. L'épuisement croissant du système immunitaire porte au syndrome d'immuno-déficience secondaire, avec les leucocytes polymorphônucléaires profondément supprimés. Les auteurs ont étudié 27 patients en réalisant en tout 221 tests. Les résultats montrent que le chimiotactisme était significativement plus haut chez les patients survivants que chez les patients décédés. Il est possible que le superoxideanion et l'eau oxygénée jouent un certain rôle dans la pathogénie du syndrome de détresse respiratoire adulte. Cependant, la production d'eau oxygénée après la stimulation avec la formyl-méthionyl-leucyl-phénylalanine (FMLP) chez les patient décédés était presque trois fois plus que chez les patients survivants. Après la stimulation avec la FMLP les patients avec un cours clinique régulier montraient une génération de superoxideanion plus basse que les patients décédés. Les résultats indiquent que les patients décédés avaient un chimiotactisme réduit, tandis que la génération des radicaux d'oxygène potentiellement nuisibles était significativement augmentée par rapport aux patients avec un cours clinique régulier.

BIBLIOGRAPHY

1. Alexander JW., Diogigm R., Meakins J.L.: Periodic variation in the antibacterial function of human neutrophils and its relationship to sepsis. Ann Surg, 173: 206-13, 1971.
2. Alexander JW., Stinnett J.D., Ogle C.K.: Alterations in neutrophil function. In: Ninnemann J.L. (Ed.): "The immune consequences of thermal injury". Williams and Wilkins, Baltimore, 1981.
3. Chenowth D.E., Roue J.G., Hugh T.: A modified method for che motaxis under agarose. J. Immunol. Methods, 25: 337-53, 1979.
4. Deitch E.A., Landry K.N., McDonald J.C.: Postbum impaired cellmediated immunity may not be due to lazy lymphocytes but to overwork. Ann. Surg., 201: 793-802, 1985.
5. Fantone J.C., Ward P.A.: Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reaction. Am. J. Path., 107: 397-418, 1982.
6. Flick M.R., Hoeffel J., Staub N.C.: Superoxide dismutase prevents increased lung vascular permeability after air emboli in unanaesthetized sheep. Fed. Proc., 983: 405, 1981.
7. Grogan J.B.: Altered neutrophil phagocytic function in burn patients. J. Trauma, 16: 734-8, 1976.
8. Hansbrough J.F., Field T.O., Gadd M.A., Soderberg C.: Immune response modulation after burn injury: T-cells and antibodies. J. Bum Care Rehabil., 8: 509-12,1987.
9. Harlan J.M., Killen P.D., Harker L.A., Striker G.E., Wright D.G.: Neutrophil-mediated endothelial injury in vitro. J. Clin. Invest., 68: 1394-1403,1981.
10. Henson P.: The immunologic release of constituents from neutrophilic leukocytes. J. Inimunol., 107: 1535-46, 1974.
11. Knaus W.A., Wagner D.P., Draper E.A., Zimmermann J.E., Bergener M., Bastos P.G., Sirio C.A., Murphy D.J., Lotring T., Damiano A., Harrell F.E.: The Apache III prognostic system. Risk prediction of hospital mortality for critically ill hospitalized adults. Chest, 100: 1619-36, 1991.
12. Messner R.P., Reed W.P., Palmer D.L.: A transient defect in leukocytic bactericidal capacity. Clin. Immunol. Immunopathol., 1: 523-32, 1973.
13. Messerschmidt 0., Langendorff H., Boraver H.: Untersuchungen Ober Kombinationsschdden. St6rungen der Antik6rperbildung bei Mdusen, die durch Verbrennungen und Ganzkbrperbestrahlungen belastet wurden. Strablentherapie, 139: 354, 1970.
14. Ninnemann J.L.: Trauma, sepsis, and the immune response. J. Bum Care Rehabil., 8: 462-67, 1987.
15. O'Gormann R.B., Feliciano D.V., Mathews R., Mathews B., Bitondo C.G., Mattox K.L., Jordan G.L.: Correlation of immunologic and nutritional status with infectious complications after major abdominal trauma. Surgery, 99: 549-56, 1986.
16. Pick E., Keisari Y.: A simple calorimetric method for the measurement of hydrogen peroxide produced by cells in nature. J. Immunol. Methods, 38: 161-70, 1980.
17. Rapaport F.T., Bachvaroff R.J.: Kinetics of humoral responsiveness in severe thermal injury. Ann. Surg., 184: 51, 1976.
18. Rittenbury M.S., Hanback L.D.: Phagocytic depression in thermal injuries. J. Trauma, 7: 523-40,1967.
19. Sacks T., Moldow C.F., Craddock P.R., Bowers T.K., Jacobs H.S.: Oxygen radicals mediate endothelial cell damage by complementstimulated granulocytes. 1. Clin. Invest., 61: 1161-8, 1978.
20. Sakai H., Daniels J.C., Beathard G.A., Lewis S.R., Lynch S.R., Ritzman S.E.: Mixed lymphocyte culture reaction in patients with acute thermal bums. J. Trauma, 14: 53-7, 1974.
21. Skornik W.A., Dressler D.P.: Lung bacteria] clearance in the burnedrat. Ann. Surg., 172: 837-43, 1970.
22. Solberg C.O., Hellum K.B.: Neutrophil granulocyte function in bacterial infections. Lancet, 2: 727-30, 1972.
23. Solomkin J.S., Nelson R.D., Chenowth D.E., Solem L.D., Simmons R.L.: Regulation of neutrophil migratory function in burn injury by complement activation products. nn. Surg., 200:742-6, 1984.
24. Tate R.M., Repine J.E.: Neutrophils and the respiratory distress syndrome. Am. Rev. Respir. Dis., 128: 552-9, 1983.
25. Tschaikowsky K., Ruegheimer E.: Expression von fMet-Leu-Phe Rezeptoren und Produktion von Sauerstoffradikalen in Granulozyten septischer Patienten. Aenesthesist, 37: 140, 1988.
26. Warden G.D., Mason A.D., Pruitt B.A.: Evaluation of leukocyte chemotaxis in vitro in thermally injured patients. J. Clin. Invest., 54: 1001-4,1974.
27. Weening R.S., Wever R., Loos D.: Quantitative aspects of the production of superoxide radicals by phagocytizing human granulocytes. J. Lab. Clin. Med., 85: 245-52,1975.
28. Weiland J.E., Davies W.B., Holter U., Mohammed J.R., Dorinsky P.M., Gadek J.E.: Lung neutrophils in the adult respiratory distress syndrome. Am. Rev. Respir. Dis., 133: 218-25,1986.
29. Weissmann G., Smolen J.E., Korchak H.M.: Release of inflammatory mediators from stimulated neutrophils. N. Engl. J. Med., 303: 27-34,1980.
30. Westaby S.: Mechanisms of membrane damage and surfactant depletion in acute lung injury. Intensive Care Med., 12: 2-5, 1986.
31. Wood G.W., Volenee F.J., Mani M.M., Humphrey L.J.: Dynamics of T-lymphocyte subpopulations and T-lymphocyte function following thermal injury. Clin. Exp. Immunol., 31: 291-7, 197 8.