ANALYTICAL

BIOCHEMISTRY

193,

38-44 (1991)

Secretion of Myeloperoxidase by Human Neutrophils Kenneth

T. Miyasaki,*

Jin-Ping

lsoforms

Song,* and A. Rekha K. Murthy’f

*Section of Oral Biology, UCLA School of Dentistry, Center for the Health Sciences, Los Angeles, California 90024, TDepartment of Medicine, Sepulveda VA Medical Center, Sepulveda, California 91343, and UCLA School of Medicine, Center for the Health Sciences, Los Angeles, California 90024

Received

October

and

2, 1990

The human neutrophil lysosomal enzyme, myeloperoxidase (MPO), exists in three major and chromatographically distinct forms, MPO I, MPO II, and MPO III. We used cation-exchange medium-pressure liquid chromatography and kinetic microenzyme assay (or spectrophotometric monitoring) to analyze the secretion of MPO isoforms by neutrophils exposed to N-formylmethionylleucylphenylalanine (FMLP), digitonin, the ionophore A23187, and serum-opsonized zymosan A (SOZ). All three MPO isomers were released into the fluid phase after neutrophils were exposed to these secretagogues. A significant proportional increase in MPO I was released when neutrophils were stimulated with SOZ. MPO I was released in higher proportions than found in the whole cell constituency when neutrophils were stimulated with FMLP + cytochalasin B, A23 187, and digitonin, but this was not statistically significant. 0 1991 Academic Press, Inc.

An important function of phagocytes, including neutrophils, is the release of antimicrobial and inflammatory substances, including the chlorin-containing enzyme myeloperoxidase (EC 1.11.1.7; MPO)’ (1). Myeloperoxidase is a 120-150 kDa glycoprotein which exhibits three major isometric forms (“isoforms”) designated MPO I, MPO II, and MPO III (2-4). An additional small form is evident in promyelocytic cell tumors (5). The three major forms from neutrophils exhibit differences in molecular weight, cationicity, hy-

i Abbreviations used: MPO, myeloperoxidase; FMLP, N-formylmethionylleucylphenylalanine; FPLC, intermediate-pressure liquid chromatography; SOZ, serum-opsonized zymosan A; PBS, phosphate-buffered saline; NHS, normal human serum; EIA, enzyme immunoassay; LDH, lactic dehydrogenase; CM, carhoxymethylated, CETAB, cetyltrimethylammonium bromide.

drophobicity, sensitivity to inhibitors, and localization within azurophil granule subpopulations (2,4). MPO I is the largest and most hydrophobic, whereas MPO III is the smallest and most cationic. The three isoforms exhibit absorbance spectra (with Soret bands at 430 nm) and &olA2so ratios which are virtually identical (3). Some investigators have reported that all the major forms are enzymatically identical (3,6,7); however, this has been disputed by others (2,8). Exocytosis of these MPO isoforms has been reported to differ, and MPO II and MPO III were observed to be exocytosed, selectively, from neutrophils pretreated with N-formylmethionylleucylphenylalanine (FMLP) and stimulated with FMLP in the presence of cytochalasin B (9). In this pioneering study, MPO isoform release was analyzed in singlet assay by cation-exchange liquid chromatography on carboxymethylcellulose using manual kinetic spectrophotometric enzyme detection. The advent of intermediate-pressure liquid chromatography (FPLC) has increased the resolution among the three MPO isoforms and has increased the speed and reproducibility of the separation (3). Kinetic microplate enzyme assays have introduced the possibility of extremely rapid analysis of small samples. The aims of this study were to use these two techniques in combination to develop a rapid and sensitive analytical method for MPO isoform measurement and to demonstrate a practical application of this method in the characterization of MPO isoform release by neutrophils in response to FMLP, the ionophore A23167, digitonin, and serumopsonized zymosan A (SOZ). MATERIALS

AND

METHODS

Leukocytes. Leukocytes and were prepared from “buffy coat” leukocyte concentrates (American Red Cross; Los Angeles, CA), by methods previously de-

38 All

Copyright 0 1991 rights of reproduction

0003-2697/91 $3.00 by Academic Press, Inc. in any form reserved.

MYELOPEROXIDASE

scribed (3). Briefly, fresh buffy coats were centrifuged, 4OOg, 30 min, 18°C. The plasma layer was aspirated and the cellular layer was placed in 2% gelatin, 0.14 M NaCl, at 37°C for 40 min. The leukocyte-enriched upper layer was collected, and after gelatin was removed by centrifugation, 3OOg, 7 min, 18”C, the cells were resuspended in 0.14 M NaCl. Erythrocytes were eliminated by brief hypotonic lysis with ice-cold distilled water and the remaining cell suspension was layered over of 55% Percoll (Pharmacia-LKB Biotechnologies; Piscataway, NJ) in 0.14 M NaCl and centrifuged, 4OOg, 30 min, 18°C. The resultant light green pellet was washed free of Percoll and resuspended in Dulbecco’s phosphate-buffered saline, pH 7.4 (PBS). The granulocytes consisted of greater than 95% neutrophils and less than 0.5% eosinophils as assessed by Wright’s stain, and are hereafter referred to as “neutrophils.” The neutrophil concentration was adjusted to 2 X lo7 cells/ml and viability assessed by trypan blue dye exclusion. Neutrophils were treated with 5 pg/ml cytochalasin B (Sigma Chemical Co.; St. Louis, MO) 5 min prior to exposure to FMLP, as indicated. Purified neutrophils and normal Secretion assay. human serum (NHS) or PBS containing CaCl, and MgCl, were mixed together and PBS was added to provide a final volume of 0.5 ml. The final concentrations were 1 X lo7 neutrophils/ml, 20% NHS (or 0.5 mM CaCl, and 1.5 mM MgCl,), and secretagogue. In some studies, neutrophils were pretreated with FMLP (Sigma), lo-* M, for 30 min, washed, and reequilibrated for 1 h prior to cytochalasin B pretreatment as described by others (9). Final secretagogue concentrations were (a) FMLP, 10e6 M and 1 X 10-l’ M and (b) zymosan A (Sigma), 2 mg/ml, (c) digitonin, l-100 pg/ml (Sigma), and (d) the ionophore A23187, 10-5-10-6 M (Sigma). Zymosan A was opsonized in the reaction mixture by including 20% NHS. The reaction mixtures were incubated at 37°C for 15 min. The mixtures were chilled on ice for 2 min, and the samples subjected to microcentrifugation, ll,OOOg, for 2 min at 4°C. Supernatants were collected and frozen at -85°C until further assay. The quantities of all reagents and cells were increased 7- to lo-fold for spectrophotometric analysis at 430 nm. Sample preparation. Supernatant fractions were thawed and applied to the column directly after 1:l dilution with 0.02 M sodium acetate, pH 4.5 (solvent A). Pellet fractions were extracted in 0.5 ml 0.5% cetyltrimethylammonium bromide in 0.02 M sodium acetate, 0.2 M NaCl, pH 4.5, using ultrasonic disruption, 10 s, 40% cycle, 8 W. Cell debris was eliminated by microcentrifugation, ll,OOOg, 2 min, and the solvent of the solubilized fraction was exchanged against solvent A using ultrafiltration. An ultrafiltration exchange ratio of at least lo5 was achieved with CF25 membrane cones (Amicon Corp.; Danvers, MA) and centrifugation at lOOOg, 15°C. All samples, both supernatant and cell pel-

ISOFORM

RELEASE

39

let extracts, were filtered prior to loading the sample loop using 0.2 pm pore, 25 mm diameter polysulfone syringe microfilters (Gelman Sciences; Ann Arbor, MI). Isoenzyme analysis. MPO isoforms in the supernatant fluid were resolved by cation-exchange FPLC on a Mono-S HR 5/5 column (Pharmacia-LKB), using a semiconcave gradient formed by solvent A and 2.0 M NaCl, 0.02 M sodium acetate, pH 4.5 (solvent B). A flow rate of 0.5 ml/min was used to generate a 20 to 80% solvent B gradient spanning 44 ml. The fraction size was 0.25 ml (0.5 min). Spectrophotometric analyses were performed with a variable-wavelength monitor (Pharmacia-LKB) set at 430 nm and adjusted to 0.02 to 0.08 AUFS. Chromatographic profiles of enzyme activity were generated, and MPO isoforms were quantified by integration. Statistical comparisons were made where appropriate using the two-tailed Student’s t test. Microplate MPO and lysozyme enzyme analysis. MPO activity was analyzed by the method of Chance and Maehly (lo), adapted for use in a kinetic microplate reader (Molecular Devices; Palo Alto, CA). Typically, 180 ~1 of 22 mM guaiacol (Sigma) in 67 mM sodium phosphate, pH 7.0, was mixed with 20 ~1 of sample (or a serial dilution) in a 96-well flat-bottomed EIAquality microplate. Serial dilutions were used routinely to detect initial reaction kinetics in any sample. The reaction was initiated by the addition of 10 ~1 of 0.67 mM H,O, using a 12-channel pipe&or. The reaction was followed kinetically for 2-20 min using the single agitation mode and taking readings every 5 to 30 s. Initial reaction rates were determined using the SoftMax program (Molecular Devices). Lysozyme was analyzed as described by Decker (11). Briefly, sample, 5-10 ~1, was mixed with 190 ~1 of a suspension of Micrococcus leisodeikticus in microtiter plates, the absorbance of the suspension at 490 nm was initially equal to about 0.6, and the assay monitored using the kinetic microplate reader set for intermittent agitation and negative kinetics. Serial dilutions were unnecessary. The LDH assay Lactic dehydrogenase (LDH) assay. of Berger and Broida (12) was used to quantify phagocyte lysis. The method was modified for microtiter analysis as follows: 100 ~1 of an NADH-pyruvate solution (disodium /3-nicotinamide adenine dinucleotide, reduced form, 1 mg/ml, in an aqueous solution of sodium pyruvate, 0.75 mM, pH 7.5) was mixed with 10 ~1 of sample in a microtiter plate and incubated at 37°C in an humidified chamber. After 30 min, 100 ~1 of 2,4-dinitrophenylhydrazine (Sigma), 2 pg/ml, in 1 N HCl was added. The reaction was terminated by the addition of 25 ~1 of 5.0 M NaOH, and read within 30 min in a microplate reader at 490 nm. RESULTS

Normal proportions cells. The proportion

of MPO isomers in intact of MPO isomers in intact cells

40

MIYASAKI,

MPO

SONG,

III 4

2-

: MPOII

:

I

j

2’ I ,,*’\

MPO

I1 -

3 -: 1 0

I

20

40

60

80

AND

MURTHY

MPO I in the intact cells was the only difference which achieved statistical significance (P < 0.01). All three isoforms of MPO were released by neutrophils exposed to the ionophore A23187 and digitonin (Table 2). Between 0.6 and 14.2% of the total cellular MPO was released under these conditions. Although it was clear that the proportion of MPO I was slightly elevated (and that of MPO III diminished) in the fluid phase after stimulation, neither of these differences were statistically significant when comparisons were made between the released and cell-associated MPO isoforms. Secretion dose-responses to FMLP and SOZ. As anticipated, the release of MPO and lysozyme in the presence of FMLP was dose-dependent and greatly potentiated by cytochalasin B (Fig. 3). Up to 20% of the total MPO and 30% of the total lysozyme was released at

FRACTION

FIG. 1. Typical pattern of MPO isomers from granules isolated from the neutrophils of a single individual. MPO isomers were separated by FPLC using a Mono-S HR 5/5 column. MPO, 38 pg, R, 0.39, was applied. Near baseline separation was achieved using a semiconcave gradient, as shown. Solvent A was 0.02 M sodium acetate, pH 4.5. Solvent B was 0.02 M sodium acetate containing 2.0 M NaCl, pH 4.5. Eluate peaks were determined spectrophotometrically by absorbance at 430 nm, 0.04 AUFS, and identities were confirmed by enzymatic activity.

varied among donors. We have observed a range of MPO isomer content in human neutrophils; generally, MPO I -CMPO II -CMPO III. In Fig. 1 is a typical chromatogram, in which the percentage of each isomer is 12, 31, and 57% for MPO I, MPO II, and MPO III, respectively. Secretion of MPO isoforms in response to FMLP, A23187, digitonin, and SOZ. All three isoforms of MPO were released by FMLP-stimulated, cytochalasin B-pretreated neutrophils (Fig. 2 and Table 1). Although the proportion of MPO I was somewhat greater, these differences were not significant at the three concentrations of FMLP tested (10e8, 10p7, and lo-” M) in the presence of cytochalasin B. In the absence of cytochalasin B, FMLP (10e7 and lop6 M) stimulated MPO isoform release in patterns which were indistinguishable from the isoform pattern of the whole cell constituency (Table 1). All three isoforms of MPO were secreted after exposure of neutrophils to the particulate stimulus, SOZ. A modest difference could be ascertained between the relative levels of secreted MPO isomers after exposure to 2 and 0.2 mg/ml SOZ in the presence of NHS in comparison to the presence of heat-inactivated NHS (Table 1). Relatively more MPO I and less MPO III was secreted in the presence of intact serum, and the increase in MPO I proportion in comparison to the proportion of

30

80 -

A

.

B

25 60 z . i E 405

0

20

20

40

60

80

100

0

20

40 60

80

0

20

40 60

80 100

100

FRACTIONS

C

0

20 40

60

FRACTIONS

FIG. 2.

80

100

FRACTIONS

MPO isomers in the supernatant fluid after stimulation of cytochalasin B-pretreated neutrophils with (A) 10-s M FMLP, (B) 10e7 M FMLP, and (C) 10-a M FMLP. Also, (D) the MPO isomers after FMLP, 10m6 M stimulation in the absence of cytochalasin B pretreatment. Peaks were determined enzymatically. The percentage of MPO released at each concentration of FMLP is shown in Fig. 3.

MYELOPEROXIDASE TABLE

ISOFORM

DISCUSSION

1

Proportion of MPO Isomers in the Fluid Phase after Stimulation of Neutrophils with FMLP with or without Cytochalasin B and Zymosan A Treated with NHS or Heat-Inactivated NHS” Fluid [FMLP] (nM)

Pretreatment*

MPO

I

MPO * + ‘-t IL f

(%)

II

MPO

5 3 3 6 2

51 52 50 57 53

III

100

13 k 3

13 rt 6

NHS + zymosan A, 2 mg/ml Heat-inact. NHS + zymosan A, 2 mg/ml

26 I!Z 2

35 + 4

39 f 5

18 + 5

33 f 3

49 f 4

13 2 1

38 k 2

51 f 3

Isomer proportion neutrophil&

16 I? 2 14 r 3 15 k 2

MPO’

1000

B B B

10 100 1000

phase

35 35 36 33 37

Cytochalasin Cytochalasin Cytochalasin None None

41

RELEASE

+ f f f +

7 1 4 2 7

in

o Reaction conditions included 5 X lo6 neutrophils in 0.5 ml PBS + 20% NHS, 37”C, end-over-end rotation, 4 rpm, 15 min. *Cytochalasin B, 5 pg/ml, 5 min, 37°C. NHS was at 20%; heat-inactivation of NHS was achieved by heating the serum to 56°C for 1 h. ’ MPO isomer ratios determined enzymatically. MPO II and MPO III values include minor MPO forms. Values represent the means and standard deviations of triplicate assays. d Unstimulated neutrophils, 5 X lOa, were extracted with 0.5% CETAB in 0.02 M sodium acetate containing 0.2 M NaCl, pH 4.7.

concentrations between lo-* and lo-’ M FMLP. The release of MPO and lysozyme as a function of zymosan A concentration also revealed an expected dose and serum dependency. More lysosomal constituents were released in the presence of cytochalasin B and FMLP than in the presence of SOZ at the concentrations tested. About 5% of MPO and 6.5% of lysozyme was released after stimulation of neutrophils with 2 mg/ml SOZ. Little cell death was observed as assessed by the release of LDH either in the presence of FMLP or SOZ. After subtraction of serum LDH (generally, these levels were negligible), the maximum LDH released by neutrophils in these conditions never exceeded 0.6% of total cell LDH. Secretion by neutrophils preexposed to FMLP. We also used a protocol of preexposing neutrophils to lo-’ M FMLP prior to cytochalasin B treatment and FMLP stimulation. The reaction was performed without NHS in the presence of 0.5 mM CaCl, and 1.5 mM MgCl, in the reaction mixture (9). This method has been reported to result in the preferential release of MPO III. Spectrophotometric analysis (using the absorbance at 436 nm) of supernatant MPO as well as pellet fractions revealed a preferential secretion of MPO I (Fig. 4). The secreted isomers exhibit increased proportions of MPO I (Table 3).

Kinetic microplate spectrophotometric methods permit the analysis of many samples rapidly and are particularly useful if the amount of sample is limited. FPLC permits the rapid separation of enzyme isomers, and may also be used with small samples in an analytical manner. We herein report the use of FPLC and kinetic microplate enzyme assays to analyze isoforms of MPO and to demonstrate that this combined methodology can be potentially useful in the analysis of cellular secretions. Specifically, we determined whether MPO isoforms were released from neutrophils in a differential manner. Using these methods, we found that in general, MPO isoform release by neutrophils reflected the MPO composition of the whole cell. That is, all three MPO isomers were released into the fluid phase, in a ratio of about 1:3:5 (MPO 1:MPO 1I:MPO III, respectively). Subtle, quantitative differences were observed between secreted MPO isomers and the MPO isomer composition of the whole cell. Usually, these differences were not statistically significant. However, neutrophils stimulated with SOZ preferentially released MPO I. There are two possible explanations for this observation. First, neutrophils may secrete MPO I differentially in response to particulate stimuli such as SOZ; or second, all isomers are released in proportion to the total cellular content, but MPO III preferentially adsorbs to the anionic surface of the particle. We believe the second explanation to be more correct and we have observed significant preferential adsorption of MPO III to certain oral microorganisms (13). It has been reported that MPO II and MPO III are preferentially released by neutrophils which were first

TABLE

2

Proportion of MPO Isomers in the Fluid Phase after Stimulation of Neutrophils with the Ionophore A23187 and Digitonina Fluid Secretagogue A23187,l @A A23187, 10 FM Digitonin, 1 pglml Digitonin, 10 pglml Digitonin, 100 aglml Isomer proportion in neutrophils’

phase

MPOb

(%)

MPO

I

MPO

II

MPO

12 14 16 15 13

4 2 2 4 3

36 362 35 35 36

+ 2 1 * 1 ? 1 k 2

52 50 49 50 51

i f * * k

10 * 3

35 i 2

Total

+ + + + f

III

cellular released

4 3 1 2 1

0.6 4.3 1.0 7.2 14.2

55 k 3

Not

MPO (9%) A * ‘f f

0.3 0.1 0.4 1.0 1.6

applicable

a Reaction conditions included 5 X lo6 neutrophils in 0.5 ml PBS containing 20% NHS, 37”C, end-over-end rotation, 4 rpm, 15 min. b Enzyme ratios were determined enzymatically. MPO II and MPO III values include minor MPO isoforms. Values represent the mean and standard deviation of triplicate assays. ’ Unstimulated neutrophils, 5 X lOa, were extracted with 0.5% CETAB in 0.02 M sodium acetate containing 0.2 M NaCl, pH 4.7.

42

MIYASAKI,

SONG,

AND

MURTHY

25

[FMLP],

nM

6-

Q

HI-NHS

[ZYMOSAN],

mg/ml

FIG. 3. Release of (A) MPO and (B) lysozyme by cytochalasin B-pretreated neutrophils, 5 X lo6 in 0.5 ml, as a function of FMLP concentration and the release of(C) MPO and (D) lysozyme by 5 x lo6 neutrophils in the presence of zymosan A. Values shown are after 15 min, 37”C, in 20% NHS; points represent the mean of triplicate assays and vertical bars represent the standard deviation.

preincubated with FMLP, lo-’ M, and subsequently treated with cytochalasin B, reequilibrated at 4°C and exposed to 10V7 M FMLP (9). In contrast to this previous report, we did not observe the selective release of MPO III. In contrast, neutrophils stimulated with FMLP + cytochalasin B, ionophore A23187, and digitonin exhibited a tendency to selectively release MPO I (albeit, this tendency was not statistically significant). Our observations were made over a range of conditions including (a) the presence and the absence of serum, (b) varying concentrations of soluble (FMLP, digitonin, and A23187) and particulate (SOZ) secretagogues, and (c) regardless of pretreatment of neutrophils with FMLP, lo-* M. Under all conditions, and using both enzymatic and spectrophotometric assays, we could not detect any biologically significant increase in MPO III

proportion in the fluid phase or depletion of MPO III from the residual cell pellet after stimulation. We believe that some of the differences between our findings and those of earlier investigators (9) may be a result of using different chromatographic and enzyme assay methods. Neither FPLC nor kinetic microplate enzyme analysis were available when the previous study was published. Mono-S FPLC offers the advantage of greater resolution, speed, and reproducibility than CMcellulose. The kinetic microenzyme assay greatly enhances the analytical power of FPLC by reducing the required sample size and increasing the assay speed. Speed is important when there is a potential for autolysis or proteolysis. Sample size is an important consideration when the experimental material is limited, as is the case for human leukocytes. Thus, whereas statisti-

MYELOPEROXIDASE

cal analysis of replicate assays was not feasible previously; in this study, from a single buffy coat, we were able to perform 30-40 replicate assays and analyze these statistically. Another methodological explanation is that different experimental conditions could account for the differences between the previous study and the present one. For example, whereas we performed our assays in Dulbecco’s PBS, the previous study used Hepes-buffered Hank’s balanced salt solution (9). Different buffers may alter the readsorption of the relatively hydrophobic MPO I to the cell surface or the retention of the relatively cationic MPO III by the proteoglycan granule matrix. We conclude that MPO isoforms released generally reflect the composition of the whole cell. However, MPO I tends to enrich within the fluid phase, and this tendency was statistically significant when a phagocytosable particle, such as SOZ, was used as a secretagogue. To be fair, we suspect that the small differences

A

:

MPO III i

.-J

I.-

0

20

40

60

80

100

ISOFORM

43

RELEASE

TABLE

3

Proportion of MPO Isomers in the Fluid Phase and Cell Pellet after Stimulation of Cytochalasin B-Pretreated Neutrophils with FMLP: The Effect of Preexposure to FMLP, lo-’

Ma

Preexposure*

FMLP

stimulation

MPO

Cell None None FMLP,

10-s

M

None FMLP, FMLP,

1O-7 1O-7

lo-’

M

FMLP, FMLP,

1O-7 1O-7

(%)

MPO

II

MPO

M M

M M

11+ 2 11 f 1 11* 1

36 + 0 34 + 3 34 + 2

53 f 3 55 * 3 54 + 3

42 f 4 41 * 3

22 f 4 23 + 2

fluids 37 + 0 35 + 4

a Reaction conditions included 7 X lo7 neutrophils in 10 ml PBS, 37”C, end-over-end rotation, 4 rpm, 15 min. ‘Preexposure to FMLP, lo-’ M for 30 min, 37”C, static, in the by washing, incubation of cells presence of Ca*+ and M%+, followed for 1 h at 4”C, and pretreatment of cells with 5 pg/ml cytochalasin B for 5 min (9). ’ Enzyme ratios determined enzymatically. MPO II and MPO III values include minor MPO forms. Values represent the means and standard deviation of triplicate assay.

observed with soluble stimuli would also become statistically significant were we to increase the number of replicate trials; however, because the differences were small, it seems unlikely that they would be biologically significant. Future studies will be aimed at determining whether the proportional enrichment of MPO I in the fluid phase can be attributed to the cationic nature of MPO III and ionic-exchange interactions which may occur with cell-associated polyanions. Regardless, it is clear that neutrophils do not selectively exocytose MPO III and also that the least likely MPO isoform to be adsorbed from secretions is MPO I. Finally, we have demonstrated that FPLC and kinetic microenzyme assays may be extremely valuable when used together in determining MPO isoform patterns. Future studies will be necessary to extend these findings to other secretagogues, such as bacteria and fungi, and to determine the significance of the MPO isoforms in inflammatory conditions. We believe that an understanding of neutrophil MPO isoform secretion can improve our ability to understand inflammatory processes and the boundaries between immunoprotection and immunopathology.

FIG. 4. pellets lo-‘M,

lation nm.

Spectrophotometric determination of MPO isomers in cell (A) after no stimulation, (B) after stimulation with FMLP, and (C) after preexposure to FMLP, lOme M, followed by stimuwith FMLP, 1Om6 M. Eluate absorbance was monitored at 430

III

pellets

Supernatant None FMLP,

I

MPO’

ACKNOWLEDGMENTS We thank Dr. Robert I. Lehrer for his critical review script. Supported by research grants from the American

of this manuFoundation

44

MIYASAKI,

SONG,

for AIDS Research (000966-7-RG), the Universitywide Task Force on AIDS Research (R89LA103), and the United States Public Health Service, National Institutes of Health-National Institute for Dental Research (DE08161). K.T.M. is a recipient of a Research Career Development Award (DE00282) from NIH-NIDR.

REFERENCES 1. Clark,

R. A. (1983)

2. Pember,

Adu.

Zrzflammation

Res. 6, 107-146.

R., and Kinkade, J. M., Jr. (1983) Arch. Biochem. Biophys. 221, 391-403. 3. Miyasaki, K. T., Wilson, M. E., Cohen, E., Jones, P. C., and Genco, R. J. (1986) Arch. B&hem. Biophys. 246, 751-764.

4. Kinkade, Spitznagel,

S. O., Shapira,

J. M., Jr., Pember, J. K., and Martin,

S. O., Barnes, K. C., Shapira, L. E. (1983) Biochem. Biophys.

R., Res.

Comm. 114.296-303. 5. Yamada, 766-771.

M., Mori,

M.,

and Sugimura,

T. (1981)

Biochemistry

20,

AND

MURTHY

6. Suzuki, K., Yamada, M., Akashi, K., andFujikura, T. (1986) Arch. Biochem. Biophys. 245,167-173. 7. Wright, J., Yoshimoto, S., Offner, G. D., Blanchard, R. A., Troxler, R., and Tauber, A. I. 1987. Biochim. Biophys. Acta 915, 68-76. 8. Wright, J., Bastian, N., Davis, T. A., Zuo, C., Yoshimoto, S., Orme-Johnson, W. H., and Tauber, A. I. (1990) Blood 75, 238241. 9. Pember, S. O., and Kinkade, J. M., Jr. (1983) Blood 61, 11161124. 10. Chance, B., and Maehly, A. C. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 2, pp. 764-775, Academic Press, San Diego. 11. Decker, L. A. (1977) Worthington Worthington Biochemical Corp.,

Enzyme Freehold,

Manual, NJ.

pp. 185-188,

12. Berger, L., and Broida, D. (1980) Sigma Technical Bulletin, No. 500. Sigma Chemical Co., St. Louis, MO. 13. Miyasaki, K. T., Zambon, J. J., Jones, C. A., and Wilson, M. E. (1987) Infect. Zmmunol. 65, 1029-1036.