Superoxide Production by Wound Neutrophils Evidence for Increased

Activity of the

NADPH Oxidase

Philip B. Paty, MD; Ronald W. Graeff, MS; StephenJ. Mathes, MD; Thomas K. Hunt, MD \s=b\ Oxygen radical secretion by neutrophils is potentiated or "primed" by extravascular migration into wounds. To define this change in responsiveness more precisely we measured superoxide production by blood and wound neutrophils from rabbits using formylmethionyl-leucyl-phenylalanine and phorbol myristate acetate as agonists. In all experiments, the time- and dose\x=req-\ dependency of superoxide secretion were the same for blood and wound neutrophils. However, wound neutrophils produced significantly more superoxide. Furthermore, the cytochrome b component of the NADPH oxidase was found in greater quantities within wound neutrophils. We conclude that priming does little to alter the requirements for activating the NADPH oxidase but does significantly increase the velocity of superoxide generation. The data suggest that alterations in the assembly and

function of the NADPH oxidase may contribute to enhanced superoxide secretion by wound neutrophils.

METHODS Animal Model and Cell Isolation Adult New Zealand white rabbits were implanted with four stain¬ less steel wire mesh cylinders under the skin of the back using ketamine hydrochloride anesthesia. Seven days later, blood and wound fluid were collected in heparinized syringes and processed in parallel as described previously. Neutrophils were isolated at room temperature by dextran sedimentation, ammonium chloride lysis of erythrocytes, and centrifugation through 56% Percoli. Frequently, cells from two or more animals were combined to provide enough cells for testing. However, in each experiment, blood and wound neutro¬ phils were taken from the same animals. Final cell suspensions were more than 95% neutrophils by stained smears and more than 98% viable by trypan blue exclusion.

Superoxide Assay

(Arch Surg. 1990;125:65-69)

migration Extravascul a r produce Superoxide pacity neutrophils secretion potentiation

into tissue can increase the ca¬ anión. This of to is known as priming of oxygen radical and is a characteristic feature of neutrophils isolated from synovial fluid,1 bronchoalveolar washings,2 peritoneal exudates,3 and wound fluid.4 Priming can also be achieved in vitro by exposure to chemotactic agents,5 arachidonate metabo¬ lites,6 macrophage products,7 and bacterial endotoxin.8 The biochemical mechanisms of priming are unclear and may not be the same for all priming agents. Priming is an important step in the neutrophil activation sequence in wounds. In a previous study comparing neutro¬ phils isolated from blood and from subcutaneous wounds in rabbits, maximum Superoxide production was increased two¬ fold in wound cells.4 To further define the nature of neutrophil priming in wounds, the current study compared blood and wound neutrophils from rabbits in time course, dose re¬ sponse, and sequential stimulation experiments using formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) as agonists. Quantisation of cytochrome b, an essential component of the NADPH oxidase, was per¬ formed by spectral analysis. Accepted for publication September 26,1989. From the Department of Surgery, School of Medicine, University of California, San Francisco. Read before the Ninth Annual Meeting of the Surgical Infection Society, Denver, Colo, April 13,1989. Reprint requests to Department of Surgery, School of Medicine, University of California, 839 HSE, San Francisco, CA 94143-0522 (Dr Paty).

production was measured in a discontinuous Superoxide assay as the Superoxide dismutase-inhibitable reduction of ferricytochrome C by 5 x 106 neutrophils in 1 mL of Hanks' balanced salt anión

solution at 37°C as described previously.4 Data were obtained in replicates of three. Cells were incubated for 30 minutes in all experi¬ ments except those studying time dependency. In dose-response experiments the median effective dose (ED^) of the agonist was determined graphically. In sequential stimulation experiments, neu¬ trophils were incubated with various doses of FMLP for 10 minutes prior to addition of cytochrome c, PMA, and Superoxide dismutase. All other stimulations by FMLP were done in the presence of cytochalasin B, 1.25 mg/L. Cytochrome c, FMLP, PMA, and cytochalasin were purchased from Sigma Chemical Co, St Louis, Mo.

Cytochrome b Spectral Analysis Cytochrome b in lysates of neutrophil suspensions was quantitated from its characteristic absorption spectrum as described previously.9 Neutrophils were suspended at 7.5 IO9 cells per liter in 3-(N-morpholino)-propanosulfonic acid, 50 mmol/L, in potassium chloride, 100 mmol/L, pH, 7.0. Cells were lysed by sonication, and membranes were solubilized by addition of sodium deoxycholate. Each sample was scanned on a double-beam spectrophotometer from 400 to 600 nm at 0.5-nm intervals. Sample cuvettes were reduced with sodium dithionite and measured in replicates of three. Reference cuvettes contained nonreduced cell suspension. The height of the gamma peak, 424 to 428 nm, was used to calculate the concentration of cytochrome b, assuming an absorption coefficient of 106 cm /mmol.

Enzyme Assays Freeze-thaw lysates of neutrophils suspended in phosphate-buf¬ fered saline at 7.5 xlO9 cells per liter were used in all assays, ß-

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Time After FMLP Stimulation, min

Fig

1 .—Cumulative Superoxide production over time following stimu¬ lation by 10 6-mol/L formylmethionyl-leucyl-phenylalanine (FMLP).

Circles indicate blood neutrophils; triangles, wound

10

»

10

9

neutrophils.

FMLP Dose, mol/L

Glucuronidase activity was measured in a colorimetrie assay of phenolphthalein release from phenolphthalein glucuronic acid (Sigma diagnostic kit 325). One unit of enzymatic activity was defined as the release of 1 µg of phenolphthalein over 1 hour at 56°C. Lysozyme activity was measured in a photometric assay of the enzymatic diges¬ tion of Micrococcus lysodeikticus as described previously.10 Activity was expressed in microgram equivalents to a standard curve pro¬ duced using purified egg white lysozyme.

Statistics SD. Data from EDa, cytochrome b, ß-glucuronidase, and lysozyme assays were compared using paired Stu¬ dent's t test. P

Superoxide production by wound neutrophils. Evidence for increased activity of the NADPH oxidase.

Oxygen radical secretion by neutrophils is potentiated or "primed" by extravascular migration into wounds. To define this change in responsiveness mor...
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