INFECTION

AND IMMUNITY,

Feb. 1979, p. 282-286

Vol. 23, No. 2

0019-9567/79/02-0282/05$02.00/0

Chemotactic Deactivation of Human Neutrophils: Possible Relationship to Stimulation of Oxidative Metabolism ROBERT D.

NELSON,".* ROBERT T. McCORMACK,3 VANCE D. FIEGEL,' MIKE HERRON,2 RICHARD L. SIMMONS,"2

AND

PAUL G. QUIE3

Departments of Surgery,' Microbiology,2 and Pediatrics,3 University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received for publication 13 October 1978

Neutrophils preexposed to high concentrations of activated complement or synthetic N-formyl methionyl peptides are inhibited in their subsequent spontaneous and chemotactic migratory responses. We have considered the possibility that a part of this nonspecific loss of migratory function may be attributable to the interaction of the leukocytes with reactive forms of oxygen deriving from the cytotaxin-induced burst of oxidative metabolic activity. For these studies we have assessed the effect of preexposure of neutrophils from patients with chronic granulomatous disease to cytotaxins on their subsequent migratory responses. We find that these responses are not altered by preexposure to either cytotaxin. Thus, there appears to be a functional relationship between deactivation and the ability of the normal neutrophil to undergo a cytotaxin-induced respiratory burst. In studies on the mechanism of leukocyte chemotaxis, Ward and Becker have observed that preincubation of rabbit peritoneal neutrophils with chemotactic products of complement activation induces an irreversible loss of their response to a subsequent chemotactic stimulus (32). They termed this phenomenon "deactivation." More recently, chemotactic deactivation of human eosinophils (34) and polymorphonuclear neutrophils (PMN) (8, 18, 21) has also been reported. Although the mechanism of deactivation remains unresolved, Ward and Becker have offered several explanations to account for this phenomenon (31). Among these, the mechanism most frequently cited involves an esterase enzyme which is activated from its proenzyme form on interaction of the neutrophil with activated complement; activation of available proesterase followed by rapid decay of labile "activatable" esterase activity during the preincubation period is thought to be the biochemical basis of deactivation (3, 31, 32). Another mechanism which may also contribute to the deactivation phenomenon may involve the cytotaxin-induced production of highly reactive species of oxygen (13, 15, 16). Autoxidative reactions attributable to excessive production of such agents have been found to alter a number of functions of PMN stimulated by other means (6, 7, 20, 26). To assess the possible relationship of deactivation to cytotaxin-mediated stimulation of oxidative metabolism, we have studied the effect of the deactivation pro-

tocol on spontaneous and chemotactic migratory functions of PMN from patients with chronic granulomatous disease. Leukocytes from such patients provide a unique opportunity to separate the effects of chemotaxis-related events from those associated with stimulation of oxidative metabolism, since PMN from such patients are deficient in their ability to produce superoxide and, therefore, other reactive forms of oxygen (5). MATERIALS AND METHODS Preparation of PMN. Blood was drawn from healthy donors and patients with the clinical diagnosis of chronic granulomatous disease in heparinized syringes, and PMN were isolated as previously described

(23).

Measurement of PMN chemotaxis and spon-

taneous migration. The chemotactic and spontaneous migratory functions of purified populations of PMN were assessed by the chemotaxis-under-agarose method as previously described (23), but with the

following exceptions. To increase migration distances and densities of the migration patterns to improve test replication, the cell number was increased to 5 x 105 per well. Furthermore, to eliminate all possibility that a rapidly developing gradient of attractant could pass the central well containing cells to influence the migration of cells on the "B" side of the migration pattern, the method for assessing spontaneous migration was altered. This function was measured instead by considering the distance of migration of cells placed in separate wells toward wells containing tissue culture medium. The chemotactic agents, zymosan-activated serum (ZAS) and cell-free supernatant from a culture of Escherichia coli (BFE), were prepared as described 282

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MECHANISM OF CHEMOTACTIC DEACTIVATION

by Ward et al. (29, 33). Both agents were used as attractants without dilution. N-formyl methionyl-

phenylalanine (F-Met-Phe) was obtained commercially (Andrulis Research Corp., Bethesda, Md.) and used as an attractant (27) at a concentration of 1.5 x lo-4 M. Deactivation methodology. Activated complement-mediated deactivation was carried out by preincubation of 6 x 106 PMN for 20 min at 370C in 0.3 ml of ZAS diluted to a concentration of 50% (vol/vol) with minimal essential medium (MEM) supplemented with glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 ,Ag/ml) (all from Grand Island Biological Co., Grand Island, N.Y.). Control populations were preincubated under identical conditions in MEM alone. Use of serum that was heated to 560C for 30 min and diluted to a 50% concentration as the control medium was observed to result in slight stimulation of the PMN migratory functions. Consequently, MEM was chosen as the appropriate control medium. Washing of the preincubated cell populations was done by first adding 3 ml of MEM to each treated population. After sedimentation of the cells by centrifugation at 200 x g for 10 min, the supernatant fluid was decanted, and the cells were resuspended in 2 ml of MEM for counting. After counting, the cells were again sedimented by centrifugation, the supernatant fluid was decanted, and the cells were resuspended in MEM for plating at a concentration of 5 x 107/ml. Deactivation mediated by F-Met-Phe (1.5 x 1O-4 M in MEM) was carried out by the same protocol described for ZAS. All tests for spontaneous migration and chemotaxis were done in triplicate. RESULTS We first assessed the effect of preexposure of PMN from a number of healthy donors to activated complement as ZAS and to F-Met-Phe on subsequent spontaneous and chemotactic migratory responses. For these studies, we chose to use 50% and 1.5 x 10-4 M concentrations of ZAS

and F-Met-Phe, respectively, since we have previously demonstrated that these agents at these concentrations are nontoxic and have comparable deactivating potentials (22). Data derived from these experiments are summarized in Table 1. In 15 separate experiments, spontaneous migration and chemotaxis of neutrophils toward ZAS were inhibited by an average of 27 and 46%, respectively, after preincubation of the neutrophils with 50% ZAS. In another 14 separate experiments, spontaneous migration and chemotaxis toward F-Met-Phe were inhibited by an average of 27 and 64%, respectively, after preincubation with 1.5 x 10-4 M F-Met-Phe. Thus, both migratory functions of neutrophils from healthy donors are consistently inhibited by these deactivation protocols. We next studied the effect of preexposure of PMN from two patients with chronic granulomatous disease (CGD) to these cytotaxins. Neutrophilic granulocytes from both of these

283

TABLE 1. Effect of deactivation protocol on migratory functions of neutrophils from healthy donors

No. of experiments

%

Depression

Treatment' CT,ZAS 46 11

CT,FMP -C

SM 27 ± 15 27 ± 11

ZAS 15 64 ± 12 F-Met-Phe 14 a Migratory responses tested include chemotaxis toward ZAS (CT,ZAS), chemotaxis toward N-formyl methionylphenylalanine (CT,FMP), and spontaneous migration (SM). Data denote mean percent depression of migratory response tested ± standard deviation. b ZAS and F-Met-Phe were at 50% and 1.5 x 1O-4 M, respectively. -, Not tested.

patients have been previously studied in our laboratories and found to be normally responsive chemotactically to ZAS but abnormally responsive to manipulations which are known to stimulate oxidative metabolism (data not shown). It has been our experience with three CGD patients, using the chemotaxis-under-agarose assay (unpublished data), and the experience of Cates and Quie with eight CGD patients, using the membrane filter method (L. Cates and P. G. Quie, personal communication), that in the absence of infectious complications the spontaneous and chemotactic migratory responses of PMN from CGD patients are within normal limits. In two experiments, PMN isolated from a CGD patient and from a control subject were preincubated in MEM, 50% ZAS, or 1.5 x 1O-4 M F-Met-Phe and washed, and their subsequent migratory functions were tested. The results of these experiments are summarized by data presented in Table 2. The untreated PMN from both CGD patients were observed to have normal spontaneous and chemotactic migratory function. Preexposure of the PMN from the healthy donors to either ZAS or F-Met-Phe significantly reduced spontaneous migratory activity and chemotactic responses to both cytotaxins. In contrast, these migratory functions of the treated PMN from the two CGD patients were either unaffected or only minimally reduced as a consequence of preexposure to these cytotaxins. DISCUSSION For the studies described which have dealt with activated complement-mediated chemotaxis and deactivation of neutrophils, we have used activated complement in the form of zymosan-activated serum. Although it is not possible to argue with absolute certainty that these influences of ZAS can be attributed to products

284

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INFECT. IMMUN.

TABLE 2. Effect of deactivation protocol on migratory functions of neutrophils from patients with chronic granulomatous disease Expt no.

1

Donor

Control Patient 1

TTreat mentb

CTZAS

% Depression

CT,FMP

% Depression

MEM ZAS FMP MEM ZAS

3.6 ± 0.2 2.0±0.1 3.1 ± 0.1 3.0 ± 0.1

44 14

8.1 ± 0.4 6.7 ±0.3 4.5 ± 0.2 4.6 ± 0.2

17 44

3.1±0.1

0

SM

2.0 ± 0.1 1.4 ±0.1 1.5 ± 0.1 1.8 ± 0.1 1.4±0.2

% Depression

30 25

4 0 4.4±0.1 NS 2.0±0.1 0 0 0 4.6±0.2 FMP 3.0±0.1 2.3 ± 0.1 4.6 ± 0.2 MEM 3.0 ± 0.1 2 Control 1.4 ± 0.2 39 33 ZAS 1.7 ± 0.1 43 3.1 ± 0.2 13 30 2.0 ± 0.1 2.6 ± 0.1 13 3.2 ± 0.1 FMP 2.2 ± 0.1 MEM 3.0 ± 0.2 5.1 ± 0.2 Patient 2 8 2.0±0.1 9 ZAS 3.0±0.1 0 4.7±0.2 NS NS 10 2.3±0.1 0 0 4.6±0.2 FMP 3.2±0.1 P = 0.05 aMigratory responses tested include chemotaxis toward ZAS (CT,ZAS), chemotaxis toward N-formyl methionylphenylalanine (CT,FMP), and spontaneous migration (SM). Data denote mean projected distance of migration in centimeters ± standard error of the mean. NS, Not significant. b Leukocytes were preincubated in MEM, 50% ZAS, or 1.5 x 10-4 M N-formyl methionylphenylalanine (FMP).

of activated complement, the complement dependency ofsuch effects of activated serum demonstrated in other reports (18, 20, 28) leads us to believe that ZAS is an acceptable substitute for the more purified complement fractions in the experiments described. Our experience that the chemotactic and deactivating functions of the serum used are not expressed if the serum is heated to 500C for 30 min before incubation with zymosan is further evidence that the effects of ZAS described in this report are complement dependent. Another question which arises with respect to our protocol is the reality of the deactivation phenomenon we have reported. One could argue that excessive carryover of cytotaxin in the final cell suspension medium could reduce neutrophil chemotaxis and spontaneous migration to mimic the deactivation phenomenon. However, we believe that the cytotaxin-mediated reduction of these migratory functions we report is attributable instead to deactivation as described originally by Ward and Becker (31) due to the following observation. When a portion of the final wash fluid is used as an attractant or as the cellsuspending medium, we find that this fluid is neither chemotactic nor inhibitory for spontaneous migration or chemotaxis of untreated cells toward optimal concentrations of multiple cytotaxins. The results we have presented (Table 1) on deactivation of normal human PMN corroborate those reported by Miller (21), Craddock et al. (8) and Issekutz and Biggar (18) and demon-

strate that complement-mediated chemotactic deactivation is not a phenomenon unique to rabbit peritoneal neutrophils. We have demonstrated, furthermore, that synthetic cytotaxin FMet-Phe, like activated complement, possesses deactivating activity (Table 1). These results corroborate those reported by Goetzel for the effect of the tetrapeptide Val-Gly-Ser-Glu on eosinophil migratory responses (12) and those by Showell et al. for the effects of N-formyl methionyl oligopeptides on lysosomal enzyme released from human neutrophils (H. J. Showell, P. H. Naccache, G. Vitkauskas, D. Williams, R. Sha'afi, and E. L. Becker, Fed. Proc. 37:1565, 1978). A relationship of deactivation to the cytotaxin-induced respiratory burst of normal human PMN is suggested by our observation that migratory functions of CGD PMN were either not altered or only minimally reduced by preexposure to ZAS or to F-Met-Phe (Table 2). Since all migratory responses of the untreated patient PMN were normal, it seems much less probable that their failure to deactivate could be due instead totally to mechanisms, such as activation and loss of an esterase enzyme activity, needed for normal migratory functions (31). Although an activatable esterase enzyme analogous to that identified in rabbit neutrophils has not been demonstrated in human PMN, it seems beyond doubt that such an enzyme must exist, and reasonably certain that the levels of its proesterase and activated forms are within normal limits in CGD PMN. Therefore, to apply the latter mech-

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MECHANISM OF CHEMOTACTIC DEACTIVATION

anism of deactivation exclusively to explain our results would require that one postulate an activatable esterase activity in CGD PMN of unique stability. Alternatively, it is possible that activation of this esterase enzyme activity may be related to stimulation of multiple neutrophil functions, including oxidative metabolism, rather than exclusively to cell migratory responses. In support of this possibility are the reported observations that other nonmigratory responses of leukocytes are associated with activatable esterase activity (10, 17, 25). Thus, in protecting rabbit neutrophils from complement-mediated deactivation by addition of aromatic amino acid derivatives, which may bind with the activatable esterase (31), Ward and Becker may have effectively blocked complement-induced stimulation of oxidative metabolism of the treated cells. In further support of our proposal, we have observed that crude E. coli-derived chemotactic factor, which neither deactivates (30) nor induces activation of significant rabbit neutrophil esterase activity (3), also fails to stimulate human neutrophil oxidative metabolism as measured by the chemiluminescence phenomenon. We have recently reported that the deactivation phenomenon may have two components (22). Preexposure of neutrophils to greater concentrations of cytotaxin results in inhibition of a subsequent spontaneous migratory response and chemotactic responses to both the deactivating and other cytotaxins: the nonspecific component. Preincubation of neutrophils with lesser concentrations of cytotaxin results in inhibition of only a chemotactic response to the deactivating cytotaxin: the specific component. The basis of the nonspecific component of deactivation remains to be determined. Our failure to observe any loss of either the spontaneous or chemotactic migratory response of CGD neutrophils after exposure to larger doses of two cytotaxins together with the oxidative metabolic dysfunction of these cells suggest that autoxidative reactions may account for at least part of the loss of these migratory functions of normal cells on preincubation with cytotaxin. The particular by-products (1, 2, 11, 19) and the mechanism by which they might influence these migratory responses remain to be identified. Furthermore, it must be remembered that still other consequences of the interaction of PMN with cytotaxin such as loss of an esterase activity (31), release of lysosomal enzymes (4, 14) or increases in volume, agglutinability or adhesiveness (9, 24) may also contribute to some degree to the nonspecific component of deactivation. ACKNOWLEDGMENTS This study was supported by Public Health Service grants

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AI 12402 from the National Institute of Allergy and Infectious Diseases, and CA 11605 and CA 23707 from the National Cancer Institute. LITERATURE CITED 1. Allen, R. C., R. L. Stjernholm, and R. H. Steele. 1972. Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem. Biophys. Res. Commun. 47:679-684. 2. Babior, B. M., R. S. Knipes, and J. T. Curnette. 1973. Biological defense mechanisms. The production by leukocytes of superoxide, a potent bactericidal agent. J. Clin. Invest. 52:741-744. 3. Becker, E. L. 1972. The relationship of the chemotactic behavior of the complement derived factors C3a, C5a and C567 and a bacterial chemotactic factor to their ability to activate the proesterase of rabbit polymorphonuclear leukocytes. J. Exp. Med. 131:376-388. 4. Becker, E. L., J. H. Showell, P. M. Henson, and L. S. Hsu. 1974. The ability of chemotactic factors to induce lysosomal enzyme release. 1. The characteristics of the release, the importance of surfaces and the relation of the release to chemotactic responsiveness. J. Immunol. 112:2047-2054. 5. Baehner, R. L. 1975. The growth and development of our understanding of chronic granulomatous disease, p. 173-195. In J. A. Bellanti and D. H. Dayton (ed.), The phagocytic cell in host resistance. Raven Press, New York. 6. Baehner, R. L., L. A. Boxer, J. M. Allen, and J. Davis. 1977. Auto-oxidation as a basis for altered function by polymorphonuclear leukocytes. Blood 50:327-335. 7. Clark, R. A., and S. J. Klebanoff. 1977. Myeloperoxidase-H202-halide system. Cytotoxic effect of human blood leukocytes. Blood 50:65-70. 8. Craddock, P. R., J. Fehr, and H. S. Jacob. 1976. Complement-mediated dysfunction in paroxysmal nocturnal hemoglobinuria. Blood 47:931-939. 9. Fehr, J., and H. S. Jacob. 1977. In vitro granulocyte adherence and in vitro margination. Two associated complement-dependent functions. J. Exp. Med. 147: 641-652. 10. Ferluga, J., G. L. Asheson, and E. L. Becker. 1972. The effect of organophosphorous inhibitors, p-nitrophenol and cytochalasin B on cytotoxic killing of tumor cells by immune spleen cells and the effect of shaking. Immunology 23:577-590. 11. Fong, K. L., P. B. McKay, J. C. Doyer, R. B. Kule, and H. Misra. 1973. Evidence that perioxidation of lysosomal membranes is initiated by hydroxyl free radicals produced during flavin enzyme activity. J. Biol. Chem. 242:7792-7797. 12. Goetzel, E. J. 1978. Regulation of the polymorphonuclear leukocyte chemotactic response by immunological reactions, p. 161-174. In J. I. Gallin and P. G. Quie (ed.), Leukocyte chemotaxis. Raven Press, New York. 13. Goetzel, E. J., and K. F. Austen. 1974. Stimulation of human neutrophil leukocytic aerobic glucose metabolism by purified chemotactic factors. J. Clin. Invest. 53: 591-599. 14. Goldstein, L. M., M. Brai, A. G. Osler, and G. Weissmann. 1973. Lysosomal enzyme release from human leukocytes: mediation by the alternate pathway of complement activation. J. Immunol. 111:33-37. 15. Goldstein, I. M., D. Roos, H. B. Kaplan, and G. Weissmann. 1975. Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. J. Clin. Invest. 56: 1155-1163. 16. Hatch, G. E., D. E. Gardner, and D. B. Menzel. 1978. Chemiluminescence of phagocytic cells caused by Nformylmethionyl peptides. J. Exp. Med. 147:182-195.

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Chemotactic deactivation of human neutrophils: possible relationship to stimulation of oxidative metabolism.

INFECTION AND IMMUNITY, Feb. 1979, p. 282-286 Vol. 23, No. 2 0019-9567/79/02-0282/05$02.00/0 Chemotactic Deactivation of Human Neutrophils: Possi...
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