Synovial Fluid Inhibits Killing of Staphylococcus aureus by Neutrophils

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1 INFECTION AND IMMUNITY, June 1983, p Vol. 4, No /83/614-7$2./ Copyright C 1983, American Society for Microbiology Synovial Fluid Inhibits Killing of Staphylococcus aureus by Neutrophils GARY L. SIMON,* HEATHER G. MILLER, AND DAVID G. BORENSTEIN Divisions of Infectious Diseases and Rheumatology, Department of Medicine, The George Washington University Medical Center, Washington, D.C. 237 Received 25 October 1982/Accepted 11 March 1983 Serum in the extracellular environment promotes neutrophil bactericidal activity apart from its opsonizing properties. We examined the effect of non-inflammatory osteoarthritic synovial fluid on serum-mediated neutrophil killing of Staphylococcus aureus. This was done to evaluate the effect of synovial fluid on neutrophil bactericidal activity independent of opsonin concentration. With an initial inoculum of 5 x 16 CFU/ml, 1.47 ±.14% bacteria survived after 12 min of incubation with 1% serum and neutrophils. In contrast, 4.7 ±.33% bacteria survived after incubation in serum plus synovial fluid (P <.1). This inhibitory effect was directly related to the concentration of synovial fluid in the incubation mixture. Increasing the concentration of synovial fluid resulted in an increased percent survival. Studies utilizing preopsonized bacteria and radiolabeled organisms demonstrated that synovial fluid did not interfere with opsonization or phagocytosis. Intracellular bactericidal activity was assayed separately from phagocytosis by utilizing a brief ingestion period followed by the removal of extracellular bacteria by either differential centrifugation or lysostaphin treatment. The reincubation of cells and associated bacteria with serum or serum plus synovial fluid revealed that synovial fluid significantly inhibited the promoting effect of serum on neutrophil bactericidal activity. After 6 min of incubation with 1% serum, 13. ± 1.2% bacteria survived, whereas 21.5 ± 2.3% survived after incubation in serum plus synovial fluid (P <.5). Superoxide production was not affected by the presence of synovial fluid. These findings suggest that the inhibitory effect of synovial fluid is due to an interaction between synovial fluid and the serum factors that promote intracellular killing. Among patients with nongonococcal septic arthritis, the most frequently isolated pathogen is Staphylococcus aureus (6, 8, 17). During the 1-year period from 1958 to 1967, Kelly and coworkers at the Mayo Clinic isolated S. aureus from the joint fluid cultures of 87% of adult patients with suppurative arthritis (8). More recently, Rosenthal et al. isolated S. aureus from 59% of 71 adults and children with pyogenic joint infections (17). The mortality rates in these two series attributable to the septic process were 15 and 7%, respectively. Considerable morbidity is also associated with S. aureus septic arthritis. Localized complications may result in the loss ofjoint mobility from ankylosis or flexion contractures. Recurrent infection and secondary osteomyelitis may also develop. Bacteremic spread with resultant endocarditis, meningitis, or localized abscess formation may further complicate the clinical course of patients with S. aureus septic arthritis. A major host defense mechanism against infection with S. aureus is phagocytic killing by 14 neutrophils. The role of synovial fluid in mediating neutrophil killing of S. aureus has not been well defined. It has been suggested that synovial fluid is less effective than serum at promoting neutrophil opsonophagocytosis, but the mechanism of decreased activity has not been elucidated. Bodel and Hollingsworth examined synovial fluid from patients with rheumatoid arthritis and demonstrated that neutrophil bactericidal activity was decreased compared with that when serum was a source of opsonins (2). Turner and co-workers found that synovial fluid from patients with a variety of arthritides was less effective than serum in promoting the phagocytosis of yeast particles by neutrophils (21). They suggested that the decreased phagocytic capacity of synovial fluid was due to a humoral defect, which was not defined. The goal of this study was to examine the mechanism whereby synovial fluid promotes phagocytic killing less effectively than does serum. The effects of synovial fluid on opsonophagocytic killing were studied in a system

2 VOL. 4, 1983 which included pooled normal human serum to insure that the concentration of opsonin was not a factor. The results of this study demonstrate that synovial fluid exerts an inhibitory effect on the serum-mediated intracellular bactericidal activity of neutrophils. (This paper was presented in part at the 39th Annual Meeting of The American Federation for Clinical Research, Washington, D.C., May 1982.) MATERIALS AND METHODS Synovial fluid. Synovial fluid was obtained by sterile arthrocentesis from the joints of seven patients with osteoarthritis. None of the patients was receiving any medications at the time of aspiration, and in no case was the joint fluid considered to be inflammatory as measured by the synovial fluid white cell count (<2, cells per ml). The synovial fluid was centrifuged to remove cellular debris, divided into samples, and frozen at -7 C within 6 min of aspiration. When synovial fluid and serum were used in combination, synovial fluid hemolytic complement activity was inactivated by warming samples to 56 C for 3 min except where noted otherwise (15). Serum. Normal human serum was obtained from five healthy volunteers by venipuncture. The samples were allowed to clot at room temperature for 6 min and centrifuged at 1, x g at 4 C for 1 min. The sera were removed, pooled, and divided into portions, which were frozen at -7 C. A portion was thawed immediately before each assay was performed. Bacteria. S. aureus (Woods 46A) was maintained on blood agar plates. For each experiment, the organism was grown to late-log phase in Trypticase (BBL Microbiology Systems, Cockeysvilie, Md.) soy broth (TSB), washed in phosphate-buffered saline, and suspended in phosphate-buffered saline at a concentration of 5 x 1' CFU/ml determined spectrophotometrically and confirmed by the pour-plate technique. Radiolabeled bacteria were prepared by incubating S. aureus with 1 p.ci of U-14C-amino acids (.1 mci/ml; New England Nuclear Corp., Boston, Mass.) in TSB. The organisms were grown to late-log phase, washed, heat killed, and suspended in phosphate-buffered saline at a concentration equivalent to 2.5 x 18 CFU/ml. Preopsonized bacteria were prepared by incubating 2.5 x 11 CFU/ml with 1%o serum or 1%o serum plus 1%6 synovial fluid at 37 C for 3 min. The opsonized bacteria were washed and suspended in phosphatebuffered saline at a concentration of 5 x 17 CFU/ml. Isolation of neutrophils. Neutrophils were obtained daily from healthy blood donors and harvested by centrifugation on Ficoll-Hypaque density gradients and sedimentation with dextran. Erythrocytes were removed by hypotonic lysis, and the neutrophils were suspended at a concentration of 17 cells per ml in Hanks balanced salt solution containing Ca2" and Mg2+ (HBSS; GIBCO Laboratories, Grand Island, N.Y.). The cell viability was examined by the exclusion of trypan blue dye. Neutrophils pretreated with synovial fluid were prepared by incubating the cells at 37 C for 15 and 6 min in 1o synovial fluid. The cells were then washed and suspended in HBSS, and bactericidal activity was examined as outlined below. INHIBITION OF INTRACELLULAR KILLING 15 Neutrophil bactericidal activity. Neutrophils, bacteria, and serum, synovial fluid, or serum plus synovial fluid were suspended in polypropylene tubes and gently rotated at 37 C. The bacteria-to-neutrophil ratio was approximately 1:1. Except where noted, the final serum and synovial fluid concentrations in the incubation mixture were 1o. Portions were sampled at various times, and the neutrophils were lysed with sterile water. The number of viable organisms was estimated by the pour-plate technique with Trypticase soy agar. The results are expressed as the percent viable organisms calculated by the formula (colony count at T/colony count at To) x 1. Phagocytosis of radiolabeled bacteria. The uptake of radiolabeled bacteria was assayed by a modification of the method of Verhoef et al. (22). Radiolabeled bacteria, neutrophils, and serum or serum plus synovial fluid were incubated at 37 C for 2 min. Phagocytosis was stopped by adding 2. ml of HBSS without Ca2" or Mg2+ to the suspension (MHS). Cell-associated bacteria were separated from non-associated bacteria by three cycles of differential centrifugation (11 x g for 1 min). The neutrophil pellets were digested with.2 N NaOH, neutralized with 3% acetic acid, and added to scintillation fluid (Aquasure; New England Nuclear Corp.). Cell-associated radioactivity was determined in a liquid scintillation counter (LS-25; Beckman Instruments, Inc., Fullerton, Calif.). The percent uptake was calculated by the formula (cpm in neutrophil pellet/total cpm) x 1. The total counts per minute represented the total radiolabeled bacteria added to the incubation mixture. Neutrophil intracellular bactericidal activity. The separation of opsonization and phagocytosis from the intracellular killing process of neutrophils was accomplished by a modification of the method of Leijh et al. (1, 11). In brief, the bacteria preopsonized with serum or serum plus synovial fluid were incubated with neutrophils for 3 min at 37 C. During this ingestion period, neither serum nor synovial fluid was present in the incubation mixture. Phagocytosis was stopped by the addition of 2. ml of ice-cold MHS. Non-cellassociated bacteria were removed by three cycles of differential centrifugation at 4 C. The cells were suspended in HBSS and 1% serum, 1%o synovial fluid, or 1% serum plus 1%o synovial fluid and incubated for 6 min at 37 C. Portions were sampled, and the number of viable organisms was determined by the pour-plate technique. The time point was defined as the time at which the bacteria were suspended in the final incubation mixture. Intracellular bactericidal activity has also been assessed by using lysostaphin treatment to kill extracellular bacteria (2). Lysostaphin was prepared by dissolving lyophilized lysostaphin (Schwarz/Mann Chemical Co., Spring Valley, N.Y.) in sterile saline at a concentration of 1, U/ml. The stock solution was stored at -2 C. Preopsonized bacteria and neutrophils were incubated at 37C for 5, 1, or 15 min. After this ingestion period, lysostaphin (2 U/ml) was added, and the mixture was incubated for 3 min, after which trypsin (.1%) was added to neutralize the lysostaphin. The cells were washed; suspended in buffer and 1%o serum, 1%o synovial fluid, or 1% serum plus 1% synovial fluid; and incubated for 6 min. The colony counts were determined before and after the final incubation.

3 16 SIMON, MILLER, AND BORENSTEIN Superoxide production. Superoxide production was measured by a modification of the method of Curnutte and Babior, utilizing the reduction of cytochrome c (5). The stimuli for neutrophil superoxide production were phorbol myristate acetate (PMA) and preopsonized S. aureus (16). Cytochrome c, PMA, and superoxide dismutase were obtained from Sigma Chemical Co., St. Louis, Mo. PMA was stored at -2 C as a stock solution in dimethyl sulfoxide at a concentration of 2 mg/ml. For each experiment, a sample was thawed and diluted in HBSS to 5 ilg/ml. Preopsonized S. aureus was prepared by incubating 1 x 19 CFU/ml with 2% pooled normal human serum at 37 C for 3 min. Cytochrome c and superoxide dismutase were dissolved in HBSS at concentrations of 1. mm and 1 mg/ml, respectively. Neutrophils (5 x 16 cells per ml) were incubated with cytochrome c (.1 mm) and PMA (5,ug/ml) or preopsonized S. aureus (5 X 17 CFU/ml) for 2 min and centrifuged at 1,5 x g to remove the cells (and bacteria), and the absorbance at 55 nm was measured on a Perkin-Elmer Coleman 55 spectrophotometer. The effect of synovial fluid on superoxide generation was assessed by adding 1o synovial fluid to the incubation mixture. In each experiment, we included a control reaction mixture from which neutrophils were absent. In some experiments, superoxide dismutase was added to the incubation mixture at a final concentration of.1 mg/ml. The results are expressed as nanomoles of superoxide produced per 5 x 16 cells per 2 min, using the extinction coefficient E55 nm = 2.1 x 14 M-1 cm-l (14). Statistical analyses were performed with a twotailed Student's t test. RESULTS The effect of synovial fluid on the neutrophil killing of S. aureus is illustrated in Fig. 1. Synovial fluid was significantly less effective than serum at promoting the neutrophil killing of S. aureus after 6 and 12 min of incubation. After 12 min, the mean percentage of organisms (plus or minus the standard error) surviving after incubation in 1% serum and neutro- co E.o co C U phils was 1.61 ±.14%, whereas 2.7 ± 2.37% survived after incubation in 1% synovial fluid (P <.5). A more striking effect was noted when serum and synovial fluid were combined. A total of 4.5 ±.47% bacteria survived after the incubation of neutrophils, bacteria, and 1% serum plus 1% synovial fluid. When compared with serum alone, this was significantly different (P <.1). The combination of serum and synovial fluid was thus antagonistic for neutrophil opsonophagocytic killing. Significantly different survival was evident after 15 min of incubation. The percentage of bacteria surviving with serum was 36.5 ± 2.4%, whereas 42.6 ± 1.23% survived with serum and synovial fluid (P <.1). These results were not related to synovial fluid complement activity since no difference was seen between heat-inactivated synovial fluid and nonheated fluid. No bactericidal activity was evident after incubation with serum, synovial fluid, or serum plus synovial fluid in the absence of neutrophils. Furthermore, the addition of 1% heat-inactivated serum to normal serum had no inhibitory effect on the serummediated killing of S. aureus. The preincubation of neutrophils with synovial fluid did not affect subsequent bactericidal activity. We found no difference in the extent of killing of S. aureus between cells preincubated in synovial fluid and cells preincubated in HBSS. Thus, it appears unlikely that the mechanism of synovial fluid inhibition of serum-mediated neutrophil killing is related to the blockade of neutrophil receptors by a component of synovial fluid. The inhibitory effect of synovial fluid on serum-mediated neutrophil killing was directly related to the concentration of synovial fluid in the incubation mixture (Fig. 2). In the presence of 1% serum, increasing the concentration of synovial fluid resulted in a progressive increase in - Serum plus synovial fluid (Heat inactivated) Serum plus synovial fluid -*- Serum... Synovial fluid P<.1 INFECT. IMMUN Time (Min) FIG. 1. Effect of synovial fluid on serum-mediated neutrophil killing of S. aureus. The number of organisms at T = was 5.1 x x 16 CFU/ml. Each point represents the mean plus or minus the standard error of 16 determinations.

4 VOL. 4, 1983 E co.cm. cn D L Concentration of Synovial Fluid in Incubation Mixture (%) FIG. 2. Effect of synovial fluid concentration on serum-mediated neutrophil killing of S. aureus. A progressive increase in the survival of S. aureus was associated with increasing concentrations of synovial fluid. All assays contained 1%o serum. Each value is the mean plus or minus the standard error of six determinations. the survival of S. aureus after 6 min of incubation. The possibility that the inhibitory effect of synovial fluid occurred at the stage of opsonophagocytosis was examined with heat-killed radiolabeled microorganisms (Table 1). These results demonstrate no difference in the extent of opsonophagocytosis whether mediated by serum or by serum plus synovial fluid. These findings indicate that serum-mediated adherence and ingestion of S. aureus by neutrophils is not affected by synovial fluid. The separation of the intracellular killing process of neutrophils from opsonization and phagocytosis was accomplished by utilizing a brief ingestion period followed by the removal of extracellular bacteria and the subsequent reincubation of the cells and associated bacteria in buffer and serum, synovial fluid, or serum plus synovial fluid (Fig. 3). In this assay, extracellular bacteria were removed by differential centrif- TABLE 1. Opsonophagocytosis of radiolabeled S. aureus % Serum % Synovial ouptace of concn fluid concn bacteriaa ± ± ± ± ± ± ± 1.2 a Each value represents the mean plus or minus the standard error for six determinations. I INHIBITION OF INTRACELLULAR KILLING 17 ugation. The percentage of bacteria surviving after 6 min of incubation in serum was 13. ± 1.2%, whereas 21.5 ± 2.3% survived after incubation in serum plus synovial fluid (P <.5). The percentage of bacteria surviving after incubation in synovial fluid without added serum was 2.9 ±.6%. When the anti-staphylococcal enzyme lysostaphin was used to remove extracellular bacteria from the incubation mixture, qualitatively similar results were obtained (Table 2). The addition of synovial fluid to the incubation mixture resulted in a significant inhibition of the promoting effect of serum on neutrophil bactericidal activity. This inhibitory effect was evident whether phagocytosis was allowed to proceed for 5, 1, or 15 min. These findings clearly demonstrate the importance of the extracellular milieu during the process of neutrophil killing. The effect of synovial fluid on the opsonization process was further investigated by utilizing microorganisms pretreated with serum or serum plus synovial fluid (Table 3). Preopsonization with serum or serum plus synovial fluid had no significant effect on the neutrophil uptake of bacteria. After a 3-min ingestion period, the number of cell-associated serum-opsonized bacteria was 2.28 x 15 ±.15 x 15 CFU/ml, whereas it was 2.39 x 1 ±+.15 x 15 for bacteria opsonized with serum and synovial fluid. Similarly, no differences in the extent of intracellular neutrophil killing were noted except when synovial fluid was used without serum. These findings further suggest that synovial fluid does not affect the fixation of opsonins on the bacterial cell surface. Synovial fluid did not affect superoxide production as measured by the reduction of cytochrome c. In the absence of synovial fluid, the extent of PMA-stimulated cytochrome c reduction was 58.8 ± 4.1 nmol/5 x 16 cells per 2 min. When synovial fluid was added, this was 61.7 ± 5.1 nmol/5 x 16 cells per 2 min. When superoxide production was stimulated by preopsonized S. aureus, the amount of cytochrome c reduced was 24.6 ± 1.7 nmol/5 x 16 cells per 2 min. With synovial fluid, this was 28.3 ± 2.5 nmol/5 x 16 cells per 2 min. Superoxide dismutase completely blocked cytochrome c reduction. In the absence of cells or stimuli, cytochrome c was not affected by the presence of serum, synovial fluid, or serum plus synovial fluid. Similar findings have been reported by Blake et al., who studied synovial fluid from patients with rheumatoid arthritis (1). DISCUSSION Synovial fluid from patients with osteoarthritis is less effective at promoting phagocytosis than is serum (21). This has been ascribed to a

5 18 SIMON, MILLER, AND BORENSTEIN INFECT. IMMUN. E c co Incubation Mixture - Buffer ---- Serum plus synovial fluid -.-Serum... Synovial fluid Co c cm co 2 1 }P< Time (Min) FIG. 3. Effect of synovial fluid on intracellular killing of S. aureus by neutrophils. The number of organisms at T = was 2.28 x x 15 CFU/ml. A significant inhibition of the promoting effect of serum was evident when synovial fluid was included in the incubation mixture. Each point represents the mean plus or minus the standard error of 1 to 14 determinations. humoral defect, possibly to decreased synovial fluid opsonins, but few data support such a hypothesis. We have examined the effect of synovial fluid on neutrophil opsonophagocytic killing independent of synovial fluid opsonic activity. This was accomplished by utilizing normal serum as a source of opsonins. The results of this study suggest that osteoarthritic synovial fluid inhibits the serum-mediated neutrophil killing of S. aureus. The physiochemical characteristics of osteoarthritic synovial fluid differ slightly from those of normal synovial fluid (4). Viscosity is mildly decreased, hyaluronic acid concentration is reduced by 37%, and synovial fluid protein concentration is increased by almost 6%. With inflammation, synovial permeability to serum proteins increases, and among patients with inflammatory arthritides, qualitatively similar but quantitatively greater changes are noted in synovial fluid properties (9). The early steps of septic arthritis are characterized by an acute inflammatory process, which should result in increased synovial permeability and greater diffusion of serum protein into the joint space. Thus, the combination of serum and synovial fluid may be more representative of intraarticular fluid in early septic arthritis than is synovial fluid alone. Phagocytic killing of microorganisms by neutrophils is often divided into three phasesopsonization, phagocytosis, and intracellular killing. The traditional role of serum factors in mediating phagocytic killing has been to maximize the ingestion process through the opsonization of bacteria. Recent studies have shown that this view is an oversimplification of a complex process. Serum factors are necessary for the optimal intracellular killing of microorganisms by neutrophils and monocytes (3, 1-13, 18, 19). TABLE 2. Effect of lysostaphin treatment on intracellular bactericidal activity Duration of %So Surviving after 6 min of incubationa in: phagocytosis or at T HBSS Serum Synovial Serum plus fluid synovial fluid ± ± ± ± 2.8 < ±.3 NDc 4.8 ± 5. ND 53.1 ± 3.3 < ±.3 ND 37.4 ± 2.7 ND 53.4 ± 3.4 <.5 a Each b value represents the mean plus or minus the standard error of six determinations. The P value compares serum with serum plus synovial fluid. c ND, Not determined.

6 VOL. 4, 1983 TABLE 3. Effect of preopsonization with serum or serum plus synovial fluid on neutrophil intracellular bactericidal activity % Bacterial survival' Bactericidal incu- after pretreatment with: bation mixture Serum plus Serum synovial fluid HBSS 33.4 ± ± 3.5 Serum 13. ± ± 1.7 Serum plus synovial 21.5 ± ± 2.5 fluid Synovial fluid ±.8 a Each value represents the mean ± the standard error for eight determinations. Both immunoglobulin G (IgG) and complement, when present in the extracellular milieu, substantially augment phagocyte intracellular bactericidal activity. When synovial fluid was added to a neutrophil opsonophagocytic bactericidal assay containing pooled normal human serum, a significant decrease in the extent of microbial killing was observed. This was independent of synovial fluid complement activity as no change in the degree of inhibition was evident when the fluid was heat inactivated. Studies utilizing preopsonized organisms showed that synovial fluid had no demonstrable effect on the opsonization process. The effect of synovial fluid on the phagocytic process, independent of neutrophil bactericidal activity, was assessed by measuring the cellular uptake of radiolabeled microorganisms. In the presence of normal serum, synovial fluid had no inhibitory effect on neutrophil phagocytosis of S. aureus. These findings indicate that the inhibitory effect of synovial fluid is not a result of the decreased ingestion of bacteria Ṫhe effect of synovial fluid on neutrophil intracellular killing, independent of opsonization and phagocytosis, was assessed by using a brief ingestion period followed by the removal of extracellular organisms by either differential centrifugation or lysostaphin treatment. Previous work by Leijh utilizing differential centrifugation has shown that this technique permits the isolation of intracellular killing from phagocytosis (1, 11). Similarly, lysostaphin treatment kills extracellular bacteria and, presumably, bacteria that are attached but not ingested (2). The results of these studies demonstrate that synovial fluid significantly inhibits the augmenting effect of serum on intracellular bactericidal activity. Synovial fluid could inhibit intracellular bactericidal activity by several possible mechanisms. Synovial fluid could be incorporated into the INHIBITION OF INTRACELLULAR KILLING 19 phagosome and directly inhibit intraphagosomal killing. This is believed to be unlikely since microscopic examination of cells after differential centrifugation after a 3-min ingestion period revealed that almost all bacteria visualized were within the confines of the cell border, suggesting that the phagosome is already formed before synovial fluid is added to the incubation mixture. Similarly, lysostaphin should kill all noningested bacteria, leaving only those which are protected within the phagosome. It seems unlikely that synovial fluid would penetrate an already-formed -phagosome and exert an inhibitory effect. Synovial fluid could exert an inhibitory effect through a direct interaction with the cell surface of the neutrophil. However, the preincubation of cells with synovial fluid did not affect subsequent bactericidal activity, suggesting that synovial fluid does not bind to the cell surface nor does it block neutrophil receptor function. Superoxide production by neutrophils leads to the formation of free radicals which can cause depolymerization of hyaluronic acid (7). Synovial fluid viscosity is decreased in inflammatory arthritis, and it is believed that this is due to hyaluronic acid depolymerization. Such a process could result in the diversion of superoxide and other oxygen moieties from bactericidal killing. However, cytochrome c reduction was not altered by incubation in synovial fluid, suggesting that little respiratory burst energy is diverted in such a manner. An alternative explanation which would account for the experimental observations is that synovial fluid directly interacts with those serum factors that promote intracellular killing and thus blocks the augmenting effect of serum. This interaction is not likely to be due to an anticomplementary effect of synovial fluid since neither opsonization nor phagocytosis is affected. The component(s) of synovial fluid which is responsible for this inhibitory effect and the mechanism of interaction with serum remain to be defined. LITERATURE CITED 1. Blake, D. R., N. D. Hall, D. A. Treby, B. Haliweil, and J. M. C. Gutteridge Protection against superoxide and hydrogen peroxide in synovial fluid from rheumatoid patients. Clin. Sci. 61: Bodel, P. T., and J. W. Hollingsworth Comparative morphology, respiration, and phagocytic function of leukocytes from blood and joint fluid in rheumatoid arthritis. J. Clin. Invest. 45: Christie, K. E., C.. Solberg, B. Larsen, A. Grov, and. 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