Dependent Chemiluminescence Response and Bacterial Susceptibility to Phagocytosis

Transcription

1 INFECTION AND IMMUNITY, Nov. 198, p /8/11-37/5$2./ Vol. 3, No. 2 Correlation Beteen Measurements of the Luminol- Dependent Chemiluminescence Response and Bacterial Susceptibility to Phagocytosis WILLIAM D. WELCH Department of Anesthesiology, University of California, Irvine, Medical Center, Orange, California The generation of chemiluminescence by phagocytosing leukocytes has been suggested to reflect concomitant microbicidal activity. Correlation beteen measurements of the chemiluminescence response and susceptibility of bacteria to phagocytosis, hoever, has not been studied. To examine and compare a range of responses in the to assays, four Escherichia coli serotypes ere chosen as test organisms ith degrees of susceptibilities to phagocytosis ranging from to 1% bacteria killed. No complete correlation beteen peak, slope, or curve area integral measurements of the chemiluminescence response and bacterial susceptibility to phagoocytosis ere found, although a correlation beteen the to assays could be made after using specific opsoniation procedures like the addition of antiserum to selected serotypes. Intrinsic differences present among the bacterial serotypes may be responsible for the observed lack of correlation beteen the to assays. Chemiluminescence (CL), or the emission of light by phagocytosing polymorphonuclear leukocytes (PMNL), is thought to result indirectly from the generation of electronically excited oxidative species. The generated oxidative species postulated to be involved in CL reactions include 2, -OH, H22, and '2 (2, 3). Addition of chemical and enymatic inhibitors of these metabolites and the enymes leading to their generation in PMNL-bacterial phagocytosis assays causes a decrease in PMNL bactericidal activity (8), suggesting their importance in microbicidal function. Several investigators have used the CL method to evaluate various serum opsonic capabilities (1, 6, 7, 1, 11) and to compare and correlate the resulting CL ith PMNL bacterial killing (12, 14). Quantitation of the CL response in these studies as determined either graphically, by peak CL, initial slope, or curve area integration. Because no previous study has explored the interrelationship beteen such measurements of CL and bacterial killing, these parameters of PMNL-bacteria interactions ere compared and analyed by using a luminol-dependent CL (LDCL) and standard pour plate bactericidal assay. MATERIALS AND METHODS Bacteria. Escherichia coli serotype 73:K92:H1 as obtained from John C. Feeley of the special pathogens laboratory, Center for Disease Control, Atlanta, Ga. E. coli serotypes O1:Kl:NM, 16:K1:NM, and O11:K nontypable ere blood culture isolates from patients at UCLA Medical Center and ere serotyped by Bertil Kaijser, Institute of Microbiology, University of Goteborg, Seden. Preparation of human PMNL and pooled human serum. Human PMNL ere sedimented from hole blood by dextran sedimentation according to the method of Boyum (4) and processed as previously described (12). For preparation of pooled human serum, equal volumes of serum obtained from six normal healthy donors ere pooled, divided into equal portions, and kept at -8 C until needed. PMNL bactericidal assay. The in vitro bactericidal assay used has been previously described and found to be a sensitive indicator of opsonic activity and bacterial killing potential of human PMNL (13). The 1-ml reaction mixture consisted of 5 x 16 to 1.2 x 16 bacterial colony-forming units (CFU) adjusted 37 by a MacFarland standard,.1 ml of pooled human serum, and 5 x 16 PMNL. The desired bacteria ere gron to log phase in nutrient broth and then ashed tice ith normal saline before use. The percentage of bacteria killed as determined by dividing the difference in CFU from tubes ith PMNL beteen and 6 min by the base-line CFU at min ith PMNL and multiplying times 1. Serum sensitivity of bacterial isolates as determined similarly in tubes ithout PMNL. LDCL LDCL as performed as recently described (11). Briefly,.5 ml of 2 x 1' PMNL per ml in Hanks buffer as added to scintillation vials containing 1.5 ml of buffer and luminol (Aldrich Chemical Co., Milaukee, Wis.) at a final concentration in the reaction vial of 1.13 x 1-7 M. LDCL as measured ith a Beckman 23 liquid scintillation spectrophotometer at ambient temperature ith a coincident circuit sitched to the rear photomultiplier tube. The reaction as done in the dark in previously dark-adapted polypropylene vials.

2 VOL. 3, 198 Preopsoniation of bacteria. A total of 19 CFU of the desired bacterial serotype ere preopsonied ith 5% pooled human serum in a total volume of.5 ml for 3 min at 37C. Preopsoniation of bacteria ith antiserum as performed as previously described (14). LDCL as initiated by the addition of 2 pl of preopsonied bacteria to the reaction mixture for a final bacteria/pmnl ratio of 2:1. Preparation of rabbit antisera. Four- to 6-kg Ne Zealand rabbits ere immunied ith live bacteria, using a minimum of to rabbits per bacterial serotype as previously described (14). Quantitation of LDCL. The initial LDCL slope from 2 to 5 min after addition of preopsonied bacteria as calculated by the linear regression leastsquares method and expressed as counts per minute per minute. Curve area integration as determined by tracing the area under each LDCL curve on standard eight paper hich as then cut and eighed. The eights are given in units of counts per 17 min. Statistical differences beteen peak LDCL, slope, and curve area integrals ere determined by the unpaired Student t test, using a Monroe 186 calculator. RESULTS Stimulation of LDCL by four E. coli serotypes. The LDCL responses of PMNL after addition of the four E. coli serotypes, 1OL:K nontypable, 73:K92:H1, 16:K1:NM, and 1: K1:NM, are shon graphically in Fig. 1. E. coli serotypes O11:K nontypable, 73:K92:H1, and 16:K1:NM all produced significant increases over background LDCL, but each shoed a different response pattern. In contrast, E. coli serotype O1:Kl:NM displayed a negligible increase in LDCL over background LDCL. 9-,,14,12,_co 48 2,. 6- a PHAGOCYTOSIS AND CHEMILUMINESCENCE 371 Correlation beteen measurements of LDCL and susceptibility to phagocytosis. To determine hether measurements of the LDCL response after addition of the four E. coli serotypes correlated ith susceptibility to phagocytosis, three parameters of the LDCL response ere compared ith phagocytosis susceptibility as determined by percentage of bacteria killed and remaining CFU at the conclusion of bactericidal experiments. E. coli 1L:K nontypable and 16:K1:NM demonstrated 97.3 and 56.6% bacteria killed, respectively, in contrast to serotypes 73:K92:H1 and 1:Kl:NM, hich shoed % bacterial killed (Table 1). Examination of the difference in CFU from the initial inoculum and the CFU present in the reaction mixture 6 min later shoed serotype 1:K1: NM ith the highest remaining CFU, folloed by 73:K92:H1, 16:K1:NM, and 1L:K nontypable. There ere approximately 7 x 16 more CFU in the O1:Kl:NM reaction mixtures than 73:K92:H1 after incubation ith PMNL. The CFU values beteen these serotypes had a P value of less than.2. LDCL peak, slope, and curve area integration ere then examined for correlation ith these degrees of susceptibilities to killing. Peak LDCL values ere not found to be greatly different beteen serotypes 1O:K nontypable and 16: K1:NM (P >.5), although 16:K1:NM peaked approximately 25 min earlier than 11: K nontypable. Serotype 73:K92:H1, % killed, but ith feer remaining CFU than O1:Kl:NM, shoed a peak LDCL intermediate beteen the )73:K92:HI au -i o. b 2-31 _ a 1 :KI:NM.a..5,B.ACKGR...OUND BACKGROUND MINUTES FIG. 1. Temporal LDCL responses of PMNL after addition offour E. coli serotypes opsonied ith pooled human serum. Points shon represent the mean of three experiments + standard deviation.

3 372 WELCH INFECT. IMMUN. TABLE 1. Comparison of the LDCL response by peak, slope, and curve area integration ith susceptibility to in vitro phagocytosis, using four E. coli serotypesa % of organ- Change in CFUb Peak LDCL Initial LDCL Integral value of area E. coli serotype isms killed in (x17) (cpm x i3) s1ope' (cpm' x 16) under LDCL curve 1 h ~~~~~~~~~~~~~~(counts/17 min) 11:K nontypable 98.4 ± ± ± ±.17 x 17 16:K1:NM 56.6 ± ± ± 3d 1.86 ±.22e 1.11 ±.6 x 17f 73:K92:H ± -.13' 78 ± 3.67 ± ±.5 x 17h O1:Kl:NM ± ± ± ±.1 x 15 a Results are mean ± standard deviation of three b experiments. Change from initial inoculum after a 6-min incubation. c From 2 to 5 min d p>.5 compared ith 11:K nontypable. 'P>.3 compared ith O11:K nontypable. f P <.5 compared ith O11:K nontypable. ' <.2 compared ith change in CFU of O1:Kl:NM. h P <.5 compared ith 11:K nontypable, P >.1 compared ith 16:K1:NM. phagocytosis-susceptible serotype O11:K nontypable (greater than 5% killed) and serotype O1:K1:NM (% killed). Comparing LDCL slopes, the phagocytosissusceptible serotypes O11:K nontypable and 16:K1:NM had different mean values, although not significantly different (P >.3), ith the latter serotype possessing a slightly higher slope value, 1.86 versus Serotype 73:K92:H1 again demonstrated an intermediate value beteen the phagocytosis-susceptible serotypes and the serotype O1:KL:NM (% killed). A third measurement of the LDCL response, curve area integration, as also compared ith the susceptibility to phagocytosis. E. coli serotype 11:K nontypable had the greatest area, folloed by 73:K92:H1 and 16:K1:NM. Although these latter to curve area integrals ere both less than that of O11:K nontypable (P <.5), they ere not statistically different from each other, ith a P value of greater than.1. The curve area integral of O1:K1:NM as approximately 1.5 orders of magnitude less than those of serotypes O11:K nontypable, 16:K1: NM, and 73:K92:H1. Increased LDCL response after opsoniation of 73:K92:H1 ith capsular antiserum over pooled human serum. To determine hether the intermediate LDCL response of serotype 73:K92:H1 could be increased to a level comparable to that of the phagocytosissusceptible serotypes O11:K nontypable and 16:K1:NM, 73:K92:H1 as opsonied ith capsular-directed antiserum (Fig. 2). The addition of capsular-directed antiserum to PMNL bactericidal experiments has been previously shon to effectively opsonie the respective bacterial strain (14) (>9% bacteria killed). The resulting LDCL response demonstrated a significant increase over pooled human serum-opsonied 73:K92:H1, ith peak, slope, and integral values of 141 ± 4 x 13, 3.69 ±.69, and 1.35 ±.1, respectively. DISCUSSION PMNL killing of bacteria is thought to depend in part and perhaps primarily on the generation of highly reactive oxidative species by the PMNL (9). CL by phagocytosing PMNL is also believed to result from the generation of similar - oxidative species, i.e, 2, 2, OH, and H22 (2, 3). Although correlation beteen CL and susceptibility to phagocytosis has been reported (12, 14), no study has yet analyed or compared various means of quantitating the CL response ith bacterial susceptibility to phagocytosis. Of the four serum-resistant E. coli serotypes examined in this study, all demonstrated different temporal LDCL responses. Analysis of various measurements of the LDCL responses, such as slope, peak, and curve area integration, and comparison ith susceptibility to phagocytosis of the serotypes studied, hoever, did not reveal a complete correlation. Surprisingly, to of the serotypes, 73:K92:H1 and O1:Kl:NM, had equal percentages of bacteria killed (%), but 73:K92:H1 gave an LDCL response intermediate beteen the to serotypes. Although no bacteria ere killed in the bactericidal assay ith either serotype 73:K92:NM or O1:Kl:NM, i.e., the final CFU ere greater than the starting CFU, bactericidal assay experiments ith 73: K92:H1 ere found to have significantly feer CFU remaining than in experiments ith 1: K1:NM, suggesting that serotype 73:K92:H1 is more susceptible than O1:K1:NM to phagocytosis. Variation in replication time may exist among E. coli serotypes but probably could not account for the observed large difference in CFU

4 VOL. 3, 198 PHAGOCYTOSIS AND CHEMILUMINESCENCE F x C.) 12[ 73:K92:HI PLUS 16:K92:NM ANTISERUM \1 (98.3 t 1.5) 1 C,) I- Uẇ. J -j D 81-6 F _ 73:K92:HI (.),,,,/ I MINUTES FIG. 2. Increase in LDCL after addition of the capsular-directed antiserum (anti-16:k92:nm)-opsonied E. coli 73:K92:H1 over pooled human serum-opsonied 73:K92:H1. Numbers in parentheses represent mean percentage of bacteria killed in 1 h ± standard deviation of three experiments. Points on curves are mean ± standard deviation of three experiments. after a 6-min incubation period. A greater susceptibility to phagocytosis of serotype 73:K92: H1 over 1:Kl:NM may then explain the respective increase in LDCL seen ith 73:K92: H1 over Oi:Kl:NM. CL elicited by PMNL stimulated ith opsonied bacteria is most likely the result of bacteria-pmnl membrane interactions leading to the production of microbicidal oxidative species. The nature of this membrane interaction may be due to specific perturbations of the PMNL membrane via Fc, C3b, or C5a receptors or from direct contact ith bacterial outer membrane components. Previous reports have documented the increased production of 2 by human PMNL after exposure to heat-aggregated immunoglobulin G (IgG), a C3b receptor stimulus, C5a (5), and generation of LDCL ith aggregated IgG (N. J. Doll et al., Fed. Proc. 39:673, 198) and IgG-coated bacteria in the absence of heat-labile serum factors (14). Serotypes 73: K92:H1, i1:k nontypable, and 16:K1:NM may thus be more prone to successful PMNL membrane interaction than O1:Kl:NM because (i) more antibody may be bound and available for appropriate PMNL membrane contact, (ii) greater amounts of C3b may be attached, or (iii) the outer membrane structure of serotypes 73: K92:H1, 11:K nontypable, and 16:K1:NM may be more conducive to interaction ith the 1.1, IIu.-a, PMNL membrane, possibly due to their large degree of hydrophobicity compared ith the 1: K1:NM outer membrane. Although 73:K92:H1 shoed an LDCL response greater than that of 1:Kl:NM and background LDCL levels, the LDCL response as measured by peak, slope, and curve area integration as significantly less than that of the phagocytosis-susceptible serotype 1O:K nontypable (>9% bacteria killed). Hoever, addition of specific capsular antiserum resulted in a greatly increased LDCL response (Fig. 2), even higher than the LDCL response seen ith 11: K nontypable, hen comparing slope and peak LDCL measurements. The use of this capsular antiserum has previously been shon to generate increased LDCL over pooled human serumopsonied bacteria and to promote effective killing of serotype 73:K92:H1 by human PMNL (>9% bacteria killed) (14). Thus, the effect of specific opsonic procedures on certain bacterial strains can be correlated ith bacterial susceptibility to phagocytosis and ith slope, peak, and curve area integration measurements of the resulting LDCL response. In conclusion, the results do not sho a complete correlation beteen the slope, peak, and curve area integration measurements of the LDCL response and bacterial susceptibility to phagocytosis. Inherent differences among the

5 374 WELCH four E. coli serotypes studied may account for this lack of correlation. Hoever, greater bacterial susceptibility to phagocytosis as achieved by opsoniation ith antiserum (or other methods) may be correlated ith respective increases in slope, peak, and curve area integration measurements of the LDCL response. ACKNOWLEDGMENT I thank Marie Lipot for excellent secretarial assistance. LITERATURE CITED 1. Allen, R. C Evaluation of serum opsonic capacity by quantitating the initial chemiluminescent response from phagocytosing polymorphonuclear leukocytes. Infect. Immun. 15: Allen, R. C Reduced, radical, and excited state oxygen in leukocyte microbicidal activity, p In J. T. Dingle and P. J. Jacques (ed.), Lysosomes in biology and pathology. North Holland Biomedical Press, Amsterdam. 3. Allen, R. C., R. L. Stjerhnholm, and R. H. Steele 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: Boyum, A Separation of blood leukocytes, granulocytes and lymphocytes. Tissue Antigens 4: Goldstein, I. M., D. Roos, H. B. Kaplan, and G. Weissman Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. J. Clin. Invest. 56: INFECT. IMMUN. 6. Hemming, V. G., R. T., Hall, P. G. Rhodes, A.. Shigeoka, and H. R. Hill Assessment of group B streptococcal opsonins in human and rabbit serum by neutrophil chemiluminescence. J. Clin. Invest. 58: Hill, H. R., N. A. Hogan, J. F. Bale, and V. G. Hemming Evaluation of nonspecific (alternative pathay) opsonic activity by neutrophil chemiluminescence. Int. Arch. Allergy Appl. Immunol. 53: Johnston, R. B., B. B. Keele, H. P. Mirsa, J. E. Lehmeyer, L. S. Webb, R. L. Baehner, and K. V. Rajagopalan The role of superoxide anion generation in phagocytic bactericidal activity. J. Clin. Invest. 55: Klebanoff, J. S Antimicrobial mechanisms in neutrophillic polymorphonuclear leukocytes. Semin. Hematol. 12: Repine, J. E., S. F. Davies, J. R. Hoidal, and P. S. Friend Effect of C2-deficiency on the bactericidal activity and chemiluminescence response of human neutrophils in vitro. Chest 75:S252-S Stevens, P., D. J. Winston, and K. Van Dyke In vitro evaluation of opsonic and cellular granulocyte function by luminol-dependent chemiluminescence: utility in patients ith severe neutropenia and cellular deficiency states. Infect. Immun. 22: Stevens, P., and L. S. Young Quantitative granulocyte chemiluminescence in the rapid detection of impaired opsoniation of Escherichia coli. Infect. Immun. 16: Welch, W. D., W. J. Martin, P. Stevens, and L S. Young Relative opsonic and protective activities of antibodies against K1, and lipid A antigens of Escherichia coli. Scand. J. Infect. Dis. 11: Welch, W. D., and P. Stevens Serum requirements necessary for the opsonophagocytosis of Escherichia coli 73:K92:H1. Curr. Microbiol. 2: