Decreased neutrophil respiratory burst on exposure to cobalt-chrome alloy and polystyrene in vitro

Decreased neutrophil respiratory burst on exposure to cobalt-chrome alloy and polystyrene ifi vitro

Arun Shanbhag, Jun Yang, Jack Lilien,* and Jonathan Black' Departments of Bioengineering and *Biological Sciences, Clemson University, Clemson, South Carolina 29634

The effect of biomaterials on the superoxide-producing ability of neutrophils was studied. Human peripheral blood neutrophils were incubated with cobalt- base alloy (F-75) or polystyrene beads of a nonphagocytosable size. Respiratory burst activity was studied by measuring superoxide dismutase inhibitable reduction of cytochrome C. Neutrophils were found to release no more superoxide anion on incubation for up to 3 h with either material in a protein-free medium than controls without foreign material. However, the ability of neutrophils incubated with either biomaterial to subsequently respond to phorbol myristate acetate challenge

was decreased (p < 0.05). Chemical analysis of supernatants for the F-75 samples showed a high concentration of cobalt in the medium within 1 h of incubation. Minimal chromium and nickel was detected. No correlation could be demonstrated between metal in solution and the respiratory burst defect in neutrophils. Instead it appears that interaction of cells with either surface was the critical event in altering the response to phorbol myristate acetate. This observed functional defect may play a n important role in rendering tissue around implanted biomaterials susceptible to infections.

IN T RODUC T ION

Surgeons recognize that patients with artificial implants are predisposed to infection at the operative site.' Once established, implant site infections are very resistant to drug therapy and the only safe course is to remove the implant and debride the site of Three general hypotheses have been advanced to explain how foreign bodies (FB) act to promote infection': (a) Damage to host (b) physical protection of the bacteria by the foreign body9-" and (c) impairment of host defen~e. '~- '~

The third hypothesis implies that host defense mechanisms (phagocytic function included) may be impaired by either direct toxicity of implant material to phagocytic cells and/or suppression of neutrophil he mot ax is.'^ Neu- trophils exposed to poly-(amide) (Nylon) fibers, glass beads and poly-(methyl) methacrylate in vitro have decreased bactericidal ability and a reduced cellular enzyme Furthermore, neutrophils in exudates from around implanted poly-(tetrafluoro) ethylene (Teflon) cages have been shown to be deficient in phagocytic and bactericidal ability.'4,'s

'To whom correspondence should be addressed

Journal of Biomedical Materials Research, Vol. 26, 185-195 (1992) 0 1992 John Wiley & Sons, Inc. CCC 0021-9304/92/020185-11$4.00

186 SHANBHAG ET AL.

This study also examines the effect of biomaterials on an active cellular defense mechanism: neutrophil function. We have analyzed the effect of short- term contact (3 h) with biomaterials on human neutrophil respiratory burst potential and consequent production of superoxide anion (SOA). Selecting both a metallic and nonmetallic material permitted us to study the effects of corrosion products.

MATERIALS A N D METHODS

The experiment was conducted in two parts. In the first part (activation analysis) neutrophils were incubated with various foreign materials (FB) to compare the magnitude of respiratory burst produced. The experimental materials were a common cobalt-base alloy and polystyrene, both in bead form. Phorbol myristate acetate (PMA), a soluble activation stimulus, was used as a positive control, and resting cells in buffer (no FB) provided a negative control. Extracellularly released SOA in the medium was measured as reduced cytochrome C.17 To ensure that a portion of the SOA was not internalized in the phagosome, cytochalasin B treatment was conferred on all experimental as well as control groups. This cytoskeletal altering agent prevents phagosome formation but does not affect normal cellular adheren~e.'~-'' Metal concentration of postincubation media was measured by graphite furnace atomic ab- sorption spectroscopy (GFAAS).

In the second part (defect studies), after timed incubation periods with these same two materials, neutrophils were challenged by the addition of PMA to provide a maximal respiratory burst activity and the residual SOA producing capability of the neutrophils was measured.

Biomaterials

The two biomaterials used were: Cobalt-base alloy (ASTM F-75) (passi- vated: ASTM F-86) beads 160 +_ 21 pm diameter. (Zimmer Inc., IN) and polystyrene beads, 109 +_ 6 pm diameter (Polysciences Inc., PA). The amount of challenge material was chosen on the basis of the specific area of the material. We estimated the surface area of an aliquot of neutrophils and to provide every cell an opportunity to contact the materials, an areal ratio of 1:l was chosen [Fig. l(a)]. Human neutrophils are -10 pm diameter; thus each aliquot of 2.5 x 10' cells provided an approximate surface area of 7.8 x lo8 pm2. To match this area, either 176.2 mg of F-75 beads (specific area = 44.6 x los pm2/mg) or 14.9 mg of polystyrene beads (specific area = 526 X lo5 pmZ/mg) were added to each aliquot of neutrophils. Note that the condi- tion selected is that before the cells actually adhere to the surface. After adherence [Fig. l(b)], the ratio changes to 1 adhered cell surface area to twice the material area available. Both materials were gas-sterilized (ethylene oxide) and thoroughly degased before use.

NEUTROPHIL RESPIRATORY BURST 187

(4 (b) Figure 1. Cel1:material areal ratios, (a) before cell attachment, (b) after cell attachment.

Human subjects

Institutional review board approval was obtained for this study. Subjects (n = 5) were healthy males between the ages of 22 and 29 who did not have any implanted foreign materials or any ongoing infection (as indicated by normal hemoglobin and neutrophil counts). After informed consent was obtained, venous blood (100 mL/subject) was drawn in sterile polypropylene monovettes (Sarstedt, FRG) containing Acid citrate dextrose (107 mM Trisodium citrate, 128 mM D-glucose, pH 5.0) with a dilution ratio (after col- lection) of 1 : 10.

Reagents

Percoll (Sigma) was mixed with physiological salts, trisodium citrate, and human albumin (fraction V, Sigma) to adjust the density to 1.077 g/cc.’* Cy- tochalasin B (5 pg) (Sigma) dissolved 1 mg/mL in Dimethyl sulfoxide (DMSO), and diluted to 100 pg/mL in HBSS was added to each aliquot of cell suspension. Cytochrome C (Sigma) was dissolved to 30 mg/mL in HBSS and 1.5 mg was added to each aliquot. PMA (Sigma) was dissolved to 2 mg/mL in DMSO, diluted in HBSS to 20 pg/mL and 1 pg used to stimulate 2.5 X lo6 cells.22 Superoxide dismutase (SOD) (Sigma) dissolved in deionized water was used to obtain specificity in each case.

Purification of neutrophils

Neutrophils were separated by centrifugation over Percoll (1.077 g/cc, lOOOg, 25”C).I8 Contaminating red cells were lysed with Ammonium chloride buffer (155 mM NH,Cl, 10 mM KHC03, 0.1 mM EDTA; pH 7.4) at 4°C. The neutrophils were washed with PBS supplemented with 1 mg/mL D-Glucose and 5 mg/mL human albumin, and incubated in HBSS with 25 mM HEPES (without Ca++, Mg”) supplemented with 10% autologous plasma for 30 min at 37°C. Cells were rewashed with Ca++ and Mg++ free HBSS and resus- pended in HBSS with 25 mM HEPES (with Ca++ and Mg”, no phenol red). Cell count and viability were assessed by Trypan blue dye exclusion. Viabil- ity was always greater than 97% at start of the assays. Cell purity as assessed by Wright’s stain was greater than 99% polymorphonuclear leukocytes.

188 SHANBHAG ET AL.

Superoxide anion production assay

All assays for cells from each subject were conducted at the same time. At time zero either PMA, metal beads, polystyrene beads, or only carrier HBSS were added to 500 pL of cytochalasin B treated (5 min, 37°C) neutrophil suspension (5.0 x lo6 cells/mL). Cytochrome C was added to all and 90 units SOD were added to selected microt~bes.’~ The vials were gently mixed and incubated in a shaking water bath for 1 or 3 h.

For activation assays, incubation was stopped after 1 or 3 h by immersing vials in water at 4°C and SOD added to quench further production of SOA. Samples were centrifuged (400g, 5 min, 4”C), supernatant transferred to dis- posable polystyrene cuvettes (Fisher) and absorbance read at 550 nm (Spec- tronic 510, Milton Roy) [schematic in Fig. 2(a)]. Metal content in the supernatant was determined by GFAAS (see below).

For the defect studies, the experiment was performed as before until the incubation step. After 1 or 3 h incubation, 1 pg PMA was added to all vials, further incubated for l h and absorbance measured as before [Fig. 2(b)]. Absorbances for samples with SOD were subtracted from remaining samples in the same category. Three replications were made for each treatment-time combination. The entire experiment was repeated in the five volunteers.

Absorbance values were converted to nanomoles of superoxide produced per 2.5 x lo6 neutrophils. SOA generation was calculated on the basis that a change in absorbance of 1.0 at 550 nm, corresponds to the presence of 47.4 nmol of S0A.I9

Determination of metal

GFAAS was performed with a Varian model 400 spectrometer fitted with a GTA-96 furnace and Zeeman background correction. Analysis for Co, Cr, and Ni were performed using partition graphite tubes. Supernatant samples were analyzed directly or diluted in 0.03N HNOl as needed to be in the linear range of detection ( c 25 ng/mL). Detection limits were: Co: 0.5 ng/mL (ppb), Cr: 0.4 ppb Ni: 0.3 ppb.

Statistical analysis

The results were evaluated in a completely randomized block design to determine treatment means, with each biomaterial and time period as factors. Intergroup comparison was made with ANOVA. Student’s f-tests were used to compare differences between individual groups. Differences among means were judged significant with type I error rates of p = 0.05 and p = 0.01.

RESULTS

Phorbol myristate acetate (PMA) stimulated a greater than 10-fold production of superoxide anion (SOA) over negative controls, as expected for


(positive control)

Determine absorbance Incubate

Neutrophils -1 (supernatant) (surface control)

No materials (neg. controls)

(a)

PMA (positive control)

(surface control)

No materials neg. controls) stimulate 0 with PMA

(b)

Figure 2. arrangement of defect studies.

(a) Schematic arrangement of activation analysis, (b) schematic

healthy ne~trophils,2~ attesting to the quality of the neutrophils used in the assay (Fig. 3). Neutrophils incubated with F-75 beads, polystyrene beads or in HBSS did not display significant SOA production up to 3 h.

After incubation with the F-75 or polystyrene beads for 1 h, neutrophils re- tained the capacity to produce amounts of SOA in response to PMA compa- rable to control preparations of neutrophils similarly incubated and stimulated at time zero (Fig. 4, Table I). However, after 3 h of incubation with either F-75 beads ( p < 0.05) or polystyrene beads ( p < 0.01), stimulation with PMA resulted in 19-34% decrease in SOA produced respectively. Note that this decrease is greater with the polystyrene than with the F-75. In parallel assays neutrophil viability was determined to be >95% after 3 h incubation. Incubation of the neutrophils in polypropylene microcentrifuge tubes had no effect on their ability to produce SOA on PMA challenge.

Disproportionately higher concentrations of cobalt ( p < 0.01) were present in solution within 1 hour of incubation with F-75 beads (Table 11). This value

190 SHANBHAG ET AL.

70

0 PMA F-75 PS no FB

Figure 3. Activation analysis results.

70

0

m1t-r I 3 k S

T

PMA F-75 PS

Figure 4. Defect studies results

TABLE I Treatment Analysis of Defect Studies

no FB

PMA F-75 Polystyrene

No FB nd p < 0.05 p < 0.01 Polystyrene p < 0.01 nd F- 75 p c 0.05

Differences between time periods: 1 11 vs 3 h

PMA

Polystyrene no FB

F-75 nd

p c 0.05

nd p < 0.01

nd = not different No differences between treatments detected after 1 h (p < 0.05). After an incubation time

of 3 h the above observations were made.


TABLE I1 Metal Analysis”

c o Cr Ni

Concentration in alloy 64.35% 27.35% 0.04% Detection limit 0.5 0.4 0.3 (ppb)

Cell + media with F-75 1 h 552 ? 1.2 * 0.1, 0.9 ? 0.1 (3)*

3 h 580 t 111“ 1.6 ? 0.1, 1.0 t 0.1 (3)s PS l h 3.3 t 1.6 0.4 ? 0.1 0.7 2 0.2 ( 2 )

3 h 4.1 * 1.6 0.5 t 0.1 0.8 f 0.1 (3) NoFB 1 h 0.9 ? 1.6 0.4 2 0.1 0.7 ? 0.2 (2) (control) 3 h .2.0 ? 1.6 0.5 ? 0.1 0.7 t 0.1 (3)

Note: n = 5 except where indicated in parentheses. “Metal concentration in postincubation media, in which neutrophils were incubated with

bMean ? estimate of 9570 confidence interval. *Significantly different from controls (p < 0.01).

either F-75, polystyrene (PS), or with no foreign body (FB).

is only slightly elevated in the next 2 h. Essentially no metal ions are seen in either media incubated with polystyrene or in controls with no materials verifying that no contamination of the samples had taken place. Moderately increased amounts of chromium and nickel ( p < 0.05) were also present.

DISCUSSION

In our experiments we observed that human neutrophils on incubation with F-75 and polystyrene beads for up to 3 h are not stimulated to release significant amounts of superoxide anion into the medium. However, following incubation with either bead type, neutrophils were found to be deficient in SOA production on challenge with PMA. This defect was greater for neutrophils incubated with polystyrene than with F-75 beads. At this time we do not know if the neutrophils can recover their SOA producing ability in the chronic presence of foreign materials. However, since the half-life of neutrophils is reported to be only 6 h124 such a recovery might not be important.

Superoxide anion (02-) is formed after phagocytosis by the transiently activated ne~ t roph i l s .~~ The SOA is subsequently converted into highly reactive oxidants such as H202 and OH- which, together with the granule contents, are responsible for the observed bactericidal a ~ t i v i t y . ~ ~ , ~ ~ Measurement of neutrophil produced superoxide anion is thus an indicator of bactericidal ability and its mediation by biomaterial contact. If the material encountered by the neutrophil is too large to be engulfed, phagosome formation is prevented, resulting in the total release of SOA extracellularly in a process termed “frus- trated p h a g o ~ y t o s i s . ” ~ , ~ ~ , ~ ~ We produced this situation by exposing neutrophils to large beads (>lo0 pm) and by addition of cytochalasin B.

It has been suggested that short-term contact of neutrophils with foreign materials will cause degranulation and an accompanying respiratory burst.14,15 Klock and Bainton’ have shown that within 2 h, human granulocytes placed

192 SHANBHAG ET AL.

in contact with Nylon fibers strongly adhered to the fibers and released 30% of the granule enzymes (acid phosphatase and P-glucuronidase) in the ab- sence of cell lysis. Variable amounts of degranulation also occurred with fibers made of cotton wool, polysulfone, and Pyrex wool. Zimmerli et al.15 showed that guinea pig peritoneal exudate PMNs degranulated within 30 min on contact with Teflon fibers. These results suggested that degranulation following contact with a material will cause neutrophil fatigue and the resulting inability to adequately phagocytose and kill pathogens.

While the evidence that neutrophils degranulate on contact with a foreign material is convincing, respiratory burst activity has not been uniformly observed. Mora and Kaplan3' recently noted that short-term respiratory burst producing activity by neutrophils following foreign body contact may be material dependent. Some polymers (polyurethane, polystyrene) did not stimulate SOA production at up to 2 h of incubation, while Dacron velour and Velcro did result in respiratory burst activity. In our experiments we failed to observe any heightened respiratory activity in neutrophils contacting rela- tively smooth materials for up to 3 h, in agreement with this report.30

A complicating factor in studies with metal beads has been the solubility of the metal in the culture media. It is to be expected that metal ions will be car- ried into cells by protein complexing, pinocytosis, or merely by diffusion. There is an inclination to argue that metal ions in solution will also cause stimulation of the neutrophils resulting in SOA release. This, however, does not appear to be the case. Rae (1983)31 has demonstrated that Co2+, Cr3+, and Ni2' solutions (up to 15 ppm) did not reduce the ability of exposed human neutrophils to kill bacteria. Remes and Williams (1990)32 and Hunt and others (1990)33 have shown that Co2+ (0.5-10 ppm), Ni2+ (0.1-50 ppm) and Cr3+ (0.05- 10 ppm) were not chemotactic and did not stimulate oxygen burst nor lyso- soma1 enzyme release from human neutrophils. After 3 h we detected only about 0.6 ppm of Co and 4 ppm of Cr and Ni. In the face of this literature and our observation that the superoxide deficit is greater with PS than with F-75, it appears then that the metal ions are not obviously responsible for this decreased ability of the neutrophils.

From these studies it appears that the cellular response to materials is most critically dependent on adherence to the material surface. We have carefully controlled the surface areas to provide a 1:l surface ratio of neutrophils to materials. In addition the polystyrene used was examined by energy disper- sive x-ray analysis and found to be very clean (data not shown) and adequately washed to remove any leachables present. Thus, the defect appears to be due to steric alterations of cell surface receptors postcontact with inor- ganic surfaces. Those surface parameters which are most important have not been identified. However, critical surface tension, surface charge of the material and consequently geometry and relative (cell: implant) surface area are likely to be important. It is possible that some materials may only produce partial responses while others may result in extensive disruption of cell 'surface receptors, cell membranes and leakage of intracellular macromolecules. Careful control of foreign material surface properties and cell : material areal ratios are critical to a better understanding of cell-biomaterials interactions.

NEUTROPHIL RESPIRATORY BURST

CONCLUSION

193

Human neutrophils are not activated to release SOA on exposure to F-75 and polystyrene. The neutrophils are, however, significantly impaired in their ability to produce a respiratory burst after culture with these two materials and consequent stimulation with PMA. We also suggest that this deficit is not an obvious function of released soluble material and could be mediated by the characteristics of the material surface interacting with the cells. The observed decrease in neutrophil functional ability could contribute to the heightened infectability observed in patients with implants.

We are grateful to Debra Denman for assistance in pilot experiments, Mary Ladd for help with the blood draws, Bub Dillon for metal analysis, and Dr. P. Burroughs for the statistical analysis. F-75 was supplied by Zimmer, Inc., IN. This work was supported by a grant from the National Institutes of Health (NIH: 5R01 AR39334).

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

S. H. Dougherty, “Implant infections,” in Handbook of Biomaterials Eualu- ation: Scientific, Technical, and Clinical Testing of Implant Materials, A. F. von Recum (ed.), Macmillan Publishing, New York, 1986, pp. 276-289. T. J. Marrie, J. Nelligan, and J. W. Costerton, “A scanning and transmis- sion electron microscopic study of an infected endocardia1 pacemaker lead,” Circulation, 66, 1339-1341 (1982). A. G. Gristina, and J. W. Costerton, ”Bacterial adherence to biomaterials and tissue: The significance of its role in clinical sepsis,” J. Bone Jt. Surg., 67A, 264-273 (1985). T. S. J. Elliott, “Intravascular-device infections,” J. Med. Microbiol., 27, 161-167 (1988). J.C. Klock and D. F. Bainton, ”Degranulation and abnormal bactericidal function of granulocytes procured by reversible adhesion to nylon

D.G. Wright and J.I. Gallin, “Secretory responses of human neutrophils: Exocytosis of specific (secondary) granules by human neutrophils during adherence in uitro and during exudation in uivo,“ J. Immunol., 123, 285-294 (1979). A. McNamara and D. F. Williams, “The response to the intramuscular implantation of pure metals,” Biomaterials, 2, 33-40 (1981). R. A. Terkeltaub, D. A. Santoro, G. Mandel, and N. Mandel, “Serum and plasma inhibit neutrophil stimulation by hydroxyapatite crystals,” Arthritis Rheumatism, 31, 1081-1089 (1988). G. Peters, R. Locci, and G. Pulverer, “Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters,” J Infect. Dis., 146, 479-482 (1982). A.G. Gristina, ”Biomaterial-centered infection: Microbial adhesion versus tissue integration,” Science, 237, 1588-1595 (1987). B. Jansen, F. Schumacher-Perdreau, G. Peters, and G. Pulverer, ”New aspects in the pathogenesis and prevention of polymer-associated foreign-body infections caused by coagulase-negative staphylococci,” 1. Invest. Surg., 2, 361-380 (1989). A. Fleer and J. Verhoef, ’An evaluation of the role of surface hydropho- bicity and extracellular slime in the pathogenesis of foreign-body- related infections due to coagulase-negative staphylococci,” J. Invest.

wool,” Blood, 48, 149-161 (1976).

Surg., 2, 391-396 (1989).

194 SHANBHAG ET AL.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28. 29.

30.

31.

32.

W. Petty, ”The effect of methylmethacrylate on chemotaxis of polymorphonuclear leukocytes,” J. Bone Jt . Surg., 60A, 492--498 (1978). W. Zimmerli, F. A. Waldvogel, P. Vaudaux, and U. E. Nydegger, “Patho- genesis of foreign body infection: Description and characteristics of an animal model,” J. Infect. Dis., 146, 487-497 (1982). W. Zimmerli, P. D. Lew, and F. A. Waldvogel, ”Pathogenesis of foreign body infection: Evidence for a local granulocyte defect,” J. Clin. Invest., 73, 1191-1200 (1984). W. Petty, “The effect of methylmethacrylate on bacterial phagocytosis and killing by human polymorphonuclear leukocytes,” J. Bone I t . Surg., 60A, 752-757 (1978). E. Pick, “Microassays for superoxide and hydrogen peroxide production and nitroblue tetrazolium reduction using an enzyme im- munoassay microplate reader,” Methods Enzymol., 132, 407-421 (1986). D. Roos and M. de Boer, “Purification and cryopreservation of phagocytes from human blood,” Methods Enzymol., 132, 225-243 (1986). D. R. Absolom, “Basic methods for the study of phagocytosis,” Methods Enzymol., 132, 95-180 (1986). S. E. Malawista, J. B. L. Gee, and K.G. Bensch, ”Cytochalasin B revers- ibly inhibits phagocytosis: Functional, metabolic, and ultrastructural effects in human blood leukocytes and rabbit alveolar macrophages,” Yale I . Bid. Med., 44, 286-300 (1971). P. Davies, R. I. Fox, M. Polyzonis, A.C. Allison, and A. D. Haswell, ”The inhibition of phagocytosis and facilitation of exocytosis i n rabbit polymorphonuclear leukocytes by cytochalasin B,” Lab. Invest., 28, 16- 22 (1973). M. Markert, P.C. Andrews, and B.M. Babior, “Measurement of 02- production by human neutrophils. The preparation and assay of NADPH oxidase-containing particles from human neutrophils,” Methods Enzymol., 105, 358-365 (1984). M. B. Grisham, T.D. Engerson, J.M. McCord, and H. P. Jones, “A com- parative study of neutrophil purification and function,” 1. lrnmunol. Methods, 82, 315-320 (1985). D. E. Kelly, R. L. Wood, and A.C. Enders, ”Blood and lymph,” Chapter 7 in Bailey’s Textbook of Microscopic Anatomy, 18th ed., Williams & Wilkins, Baltimore, MD, 1984, pp. 232-252. J. E. Smolen, ”Characteristics and mechanisms of secretion by neutrophils,” in The Neutrophil: Cellular Biochemistry and Physiology, M. 8. Hallet (ed.), CRC Press, Boca Raton, 1989, pp. 23-61. I. M. Goldstein, M. Cerqueira, S. Lind, and H. B. Kaplan, “Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface,” J. Clin. Invest., 59, 249-254 (1977). B. M. Babior, ”Oxygen-dependent microbial killing by phagocytes, I,” N. Engl. J. Med., 298, 659-668 (1978). P. Murphy, The Neutrophil, Plenum Publishing, New York, 1976, p. 179. T. Rae, “The macrophage response to implant materials- with special reference to those used in orthopedics,” CRC Crit. Rev. Biocompatibility, 2 (2), 97-126 (1986). E. M. Mora, and S. S. Kaplan, “Superoxide release by neutrophils exposed to different biomaterials,” Trans. Soc. Biomaterials, 13, 163 (1990). T. Rae, ”The action of cobalt, nickel and chromium on phagocytosis and bacterial killing by human polymorphonuclear leucocytes: its rele- vance to infection after total joint arthroplasty,” Biomaterials, 4, 175- 180 (1983). A. Remes and D.F. Williams, ”Chemotaxis and the inhibition of chemotaxis of human neutrophils in response to metal ions,” 1. Muter. Sci. Muter. Med., 1, 26-32 (1990).

N EUTROPHIL RESPIRATORY BURST 195

33. J.E. Hunt, A. Remes, and D.F. Williams, "The stimulation of neutrophil degrada t ion by metal ions," presented at Biointeractions '90, Oxford, UK, Aug. 21-23,1990,

Received November 18,1990 Accepted August 14, 1991

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Decreased neutrophil respiratory burst on exposure to cobalt-chrome alloy and polystyrene in vitro