polymorphonuclear neutrophils in vitro

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1 The effet of arylamide on human polymorphonulear neutrophils in vitro D. MANGAN AND I. S. SNYDER British Journal of Industrial Mediine, 1978, 35, From the Department of Mirobiology, West Virginia University Medial Center, Morgantown, WV 2656, USA ABSTRACT Arylamide (CH2CHCONH2), the vinyl monomer of the industrially useful polymer polyarylamide, is a reognised neurotoxin. Investigation in our laboratory indiated that, in addition to its neurotoxi effet, arylamide depressed human polymorphonulear leuoyte (PMN) hemotaxis in vitro. As geneti or hemial inhibition of PMN phagoyti funtion frequently predisposes patients to repeated baterial infetions, the in vitro effets of arylamide on several other human PMN funtions were studied. Arylamide in onentrations up to 37 5 mg/ml had no effet on trypan blue uptake. However, baterial ingestion, killing, and indued hemiluminesene were depressed by pre-treatment with arylamide (1 mg/ml). It seems unlikely that arylamide exposure alters host resistane to baterial infetions, beause (a) large doses of arylamide are neessary to interfere with phagoyti funtions, (b) arylamide reats readily with proteins on many tissue ells and may be made inaessible or non-toxi to PMNs, and () PMNs have a rapid turnover rate in the body and non-funtional ells would be rapidly replaed by funtional ells. The polymorphonulear neutrophil leuoyte (PMN) is an important nonspeifi defene against infetion. Phagoytosis of bateria, the main funtion of PMNs, an be divided into six funtional steps: hemotaxis, opsonisation (reognition), ingestion, degranulation, ativation, and bateriidal ativity. Certain drugs, suh as olhiine (Caner, 1965), steroids (Beisel and Rapoport, 1969), and ethanol (Brayton etal., 197), temporarily depress one or more PMN phagoyti funtions in vitro and are assoiated with inreased host suseptibility to baterial infetions. Similarly, an inherited defet in a partiular phagoyti funtion, suh as in oxidative metabolism in hroni granulomatous patients (Holmes et al., 1967; Quie et al., 1967), or in hemoaxis in patients with the 'lazy leuoyte syndrome' (Miller et al., 1971), dereases the ability of the PMN to remove the baterium effetively, and thereby predisposes the patient to infetion. Arylamide (CH2CHCONH2) is a reative ompound whih an readily undergo vinyl-type polymerisation to produe the industrially useful polymer, polyarylamide. As a polymer the ompound is nontoxi, but in the monomeri form arylamide is a well reognised neurotoxin (Spener and Reeived for publiation 4 November 1977 Aepted for publiation 18 January 1978 Shaumburg, 1974a). Arylamide penetrates intat skin and the effets of sub-linial exposures are umulative (Spener and Shaumburg, 1974a, 1974b). Preliminary investigations in this laboratory indiated that arylamide inhibited PMN hemotaxis in vitro, thus suggesting that arylamideintoxiated individuals may be more suseptible to baterial infetions. To follow up these findings, and beause of an inreased interest in the biologial effets of important industrial hemials, the effet of arylamide on human PMN funtions in vitro was investigated. Materials and methods ISOLATION OF PMN LEUCOCYTES Human PMNs from peripheral venous blood were olleted in 1 ml heparinised (143 U/tube) Vautainer tubes (Beton-Dikinson and Co., Rutherford, NJ). The blood was transferred to vertial sterile silionised srew-ap tubes (1 x 15 mm) and mixed with 5 ml old heparinised (1 U/ml) 3% dextran (T5, Pharmaia Fine, Pisataway, NJ) solution in -15 M NaCl and allowed to separate for 3 min at 37 C. The leuoyte-rih upper layer was arefully removed with a apillary pipette and entrifuged at 25 g for 1 min at room temperature (RT) 35

2 36 in silionised glass entrifuge tubes. Residual erythroytes were lysed by resuspension of the leuoyte pellet in 2 ml of -2% NaCl for 3 s followed by the addition of 2 ml of 1-6 % NaCl (Clark and Kimball, 1971). The leuoytes were repeatedly washed in either old saline or Hanks balaned salt solution without sodium arbonate (HBSS) and then resuspended in old saline or HBSS. In the hemiluminesene studies, HBSS without phenol red (HBSS-x) was used. Leuoyte ounts were determined with a haemaytometer, or eletronially ounted (Coulter Eletroni In., Hialeah, Florida) and adjusted to the required ell onentration with HBSS. ACRYLAMIDE Arylamide was eletrophoresis purity reagent grade and reported to be nearly 1% pure. Its major ontaminant was water, and there were no known ontaminating heavy metals or toxi ompounds (Bio-Rad, Rihmond, California, personal ommuniation). PRETREATMENT OF PMNS WITH ACRYLAMIDE Aliquots of the PMN suspension (5 x 16 to 1 x 17 PMN/ml) were added to silionised 1 ml entrifuge tubes ontaining equal volumes of various onentrations of arylamide dissolved in HBSS. Leuoytes mixed with an equal volume of HBSS served as ontrols. The tubes were gently inverted and inubated in a 35 C water bath. After 3 min the tubes were spun at 25 g for 5 min at RT. The ell pellets were washed twie in old saline and resuspended in HBSS with 1% pooled human serum to the original volume. TRYPAN BLUE EXCLUSION PMNs were examined for viability as determined by ability to exlude trypan blue. A -5 ml aliquot of the treated ell suspension was plaed in a 12 x 75 mm tube. To this, -1 ml of a -4/% solution of trypan blue in saline (Gibo, Grand Island, NY) was added, the ell suspension was mixed thoroughly and allowed to stand for 1 min at RT. The number of stained (damaged) and unstained (undamaged) leuoytes were ounted and the results expressed as perentage viable ells. PRODUCTION OF THE POSITIVE CHEMOTACTIC STIMULUS (PCS) The hemotati fragment of omplement, C5a, was generated as desribed by Ward (1976). Zymosan (1 mg) was added to fresh serum (1 ml) and inubated at 37 C for 1 h. This preparation was diluted 1:1 in HBSS for use in the hemotaxis assay. CHEMOTAXIS ASSAY D. Mangan and L S. Snyder A modified Boyden hamber (Belo Glass Co., Vineland, NJ) was used to assess PMN migration (Boyden, 1962). The hamber onsists of an upper and lower ompartment separated by a ellulose membrane with a pore size of 5,um and a thikness of about 125,tm. One ml of the PMN suspension adjusted to 2 5 x 16 ells/ml was added to the upper ompartment and 5 ml of the PCS was loaded into the lower ompartment. The hambers were inubated at 37 C for 3 h after whih the filters were washed, stained with haematoxylin, leared in xylene, inverted, and mounted on glass slides (Ward, 1976). Migration was determined by soring all ells (PMNs) whih had migrated into the filter 5,um or more from the starting side. Seven to ten random high power (x 21) fields were averaged per filter. Tripliate hambers were used for eah onentration of arylamide tested. BACTERIA The Staphyloous epidermidis strain was obtained from a departmental stok ulture held at 4 C on a nutrient agar slant. The ulture was heked for purity by subulture on to blood agar, Gram staining and biohemial testing. PMN INGESTION AND BACTERICIDAL CAPACITY Staphyloous epidermidis was grown for 18 h in 8 ml of Trypti soy broth (Difo) at 35 C. The ultures were entrifuged, the baterial pellet washed twie with old saline, and resuspended to the original volume in old saline. A 1:1 dilution of this suspension, normally yielding around 16 olonyforming units (CFU) per ml, served as the working onentration. Ingestion and bateriidal apaity of the arylamide-treated PMNs were determined by a modifiation of the methods desribed by Mandell and Hook (1969). Two ml of the PMN ell suspensions were transferred to sterile silionised 13 x 1 mm srew-ap tubes. To eah tube 1 ml of the staphylooal suspension was added. This provided a bateria: PMN ratio of approximately 1:1. Eah tube was gently inverted and a -6 ml aliquot was immediately removed and plaed in hilled sterile entrifuge tubes. This was the ' time' speimen. The leuoyte-bateria suspensions were inubated at 37 C with end-over-end rotations (Roto-rak, Fisher Sientifi Co., Pittsburgh, Pa.). At 3, 6, and 12 min, -6 ml aliquots were removed from eah tube. From eah -6 ml aliquot taken at eah time interval, O1 ml was added to a 9-9 ml sterile distilled water blank and vigolously shaken to disrupt the

3 Arylamide and PMN funtions PMNs. Viable bateria were ounted by standard dilution and pour plate methods. These ounts reptesented the total number of live bateria, both intraellularly and extraellularly, in the leuoytebateria suspension. To the remaining 5 ml of the sample aliquot, 4-5 ml old sterile saline was added and mixed gently. The tubes were entrifuged at 25 g for 5 min, after whih the supernate was assayed for viable bateria. This ount represented bateria whih were not ingested and not killed by the extraellular release of leuoyte lysosomal enzymes. The PMN pellet (approximately -1 ml) was resuspended in 4 5 ml sterile water, vigorously shaken, and the viable bateria ounted. This ount represented bateria whih had been engulfed, but not killed, and bateria whih had been trapped by PMN aggregations during entrifugation. CHEMILUMINESCENCE Human PMNs were isolated, as previously desribed, and resuspended to a onentration of 5 x 16/ml in HBSS-x. The ells were treated with arylamide, washed twie in HBSS-x, and resuspended in HBSS-x with 1% fresh autologous serum (i.e. serum from the same volunteer from whih PMNs had been obtained). Staphyloous epidermidis was grown for 18-2 h in 8 ml Trypti soy broth at 35 C. The ultures were entrifuged, washed twie in old saline and the pellet was then opsonised with 3 ml of fresh autologous serum for 3 min at 35 C. The ells were then spun at 25 g at room temperature for 5 min, washed one in HBSS-x, and resuspended in this same buffer to original volume. This yielded about 17 viable bateria per ml. Chemiluminesene (CL) was measured in a Pakard sintillation spetrometer, Model 332, operating at 4 C using the out-of-oinidene summation mode, with a window setting at -oo and 1% gain (Allen et al., 1972, 1974). All proedures were performed under dim light to minimise bakground light emission. Silionised glass sintillation vials were dark-adapted for at least 24 h before use. One ml of leuoytes was added to dupliate vials and warmed for 5 min in a 35 C water bath. The bakground CL of eah vial was measured for 2 s at 5 min intervals over a total period of 15 min. At the end of this time, 1 ml of HBSS-x was added to one of the dupliate vials ('resting ells') and 1 ml of opsonised baterial suspension was added to the other vial ('ativated ells'). The addition of the HBSS-x or bateria was staggered so that measurement of CL was at equal times from time. To measure CL, eah vial was removed from the water bath, blotted dry, the HBSS-x or bateria added, gently mixed, and 37 immediately lowered into the sintillation ounter (about 2-3 s after the addition). CL was measured for a 2 s interval and the vial was returned to the water bath. Correted ounts for eah vial were obtained by subtrating the average bakground CL during the initial 15 min period from the CL obtained after ativation of the ells with either HBSS-x or bateria. CL measurement was ontinued at 5 min intervals over a period of 45 min. Results TRYPAN BLUE EXCLUSION Human PMNs exposed to arylamide were tested for viability by trypan blue exlusion. Pretreatment for 3 h at 37 C with arylamide in onentrations as high as 37-5 mg/ml did not lead to staining of the PMNs. Under the light mirosope the PMNs treated with arylamide appeared to have normal morphology. CHEMOTAXIS The Table shows the hemotati ativity of arylamide-treated and HBSS-pretreated (ontrol) PMNs obtained during a single experiment. Arylamide in a onentration of 5 mg/ml had very little effet on the number of ells migrating toward the PCS, ompared with the ontrol ells. Arylamide onentrations of 1- and 5-mg/ml dereasedthe number of migrating ells by about one-third. Pretreatment with arylamide at a onentration of 1 mg/ml inhibited movement of 9% of the PMNs. It should be noted that hanging the onentration of arylamide five-fold (from 1 mg/ml to 5 mg/ml) did not inrease inhibition of hemotaxis above that aused by 1 mg/ml. However, a two-fold inrease (from 5 mg/ml to 1 mg/ml) inhibited hemotaxis by 9%. Whether this suggests that there are two ative omponents in the arylamide preparation, or that more than one funtion of hemotaxis is affeted, is not known. PMN INGESTION AND BACTERICIDAL CAPACITY To further define the effet of arylamide on phagoyti funtions, baterial ingestion and killing apaity of pretreated PMNs were studied Ingestion of staphylooi was dereased in PMNs pretreated with 1- mg/ml arylamide, the onentration at whih hemotaxis was severely inhibited (Fig. 1). Similar data were obtained from two separate experiments. Stained smears of the leuoytes removed at various times from the reation mixture showed intraellular bateria in both arylamideand ontrol-pretreated PMNs.

4 38 Table PMN hemotaxis after pretreatment with arylamide Arylamide Chemotati %/ inhibition onentration ativity (Mg/Mb* (ells1hpft) Control ± i i Positive hemotatant: zymosan-treated fresh human serum. *Final onentration of arylamide in the pretreatment reation mixture. tnumbers represent the mean number of ells per high power field (x 21) ± standard error of the mean, whih have migrated at least SOum into the filter. Results from three hambers. ( Time of inubation (mm) Fig. 1 Ingestion of bateria by PMNs pretreated with arylamide. The ordinate represents the number of Staphyloous epidermidis esaping ingestion after inubation with PMNs pretreated with arylamide (1- mg/ml) (A) or HBSS (ontrol) (O)for 3 mini at 35SC. These values were obtained by ounting the bateria in the supernatant remaining after removal of the PMNs by entrifugation. A diminished intraellular killing apaity was deteted by the inability of arylamide-pretreated PMNs to destroy ingested bateria. Figure 2 depits data from one of two experiments whih yielded similar results. The ontrol PMNs redued the number of ell-assoiated bateria from 4 x 14 to i 4. 3 o -Q D. Mangan and I. S. Snyder Tirne of inubation ( min) Fig. 2 Intraellular bateriidal ativity ofpmns pretreated with arylamide. The ordinate represents the number of Staphyloous epidermidis ingested but not killed by PMNs pretreated with arylamide (1- mglml) (A) or HBSS (ontrol) (O) for 3 min at 35 C. These values were obtained by sedimenting the PMNs by entrifugation. The bateria were releasedfrom the PMNs by lysis and ounted. 7 x 12 CFU/ml over the 12 min period. In omparison, the number of viable intraellular bateria in the arylamide-pretreated sample dereased from 7 x 14 to 9 x 13 CFU/ml over the same period. The total bateriidal apaity (intraellular and extraellular) of PMNs pretreated with arylamide is shown in Fig. 3. Conentrations of arylamide above 5- mg/ml depressed total bateriidal apaities ompared with the ontrol, while onentrations below 5- mg/ml inreased baterial killing. PMNs pretreated with 5- mg/ml arylamide had a killing apaity similar to the ontrol PMNs. The small inrease in the number of bateria in samples laking PMNs did not appear substantially to affet the results of the test. CHEMILUMINESCENCE CL was used as a measure of PMN oxidative metabolism during phagoytosis (Allen et al., 1972, 1974). Control PMNs, ativated with opsonised Staphyloous epidermidis, gave reproduible results with maximal CL of about 7-

5 Arylamide and PMN funtions b U).) 6._ 5 o5 -e ~ C- 9 U Tirme of inubation ( min) Fig. 3 Total bateriidal apaity of PMNs pretreated with varying onentrations ofarylamide. The ordinate represents the total viable Staphyloous epidermidis remaining after inubation with PMNs pretreated with arylamide in onentrations of 1P (), 2 5 (-), 5S (LI), 7 5 (A), and 1 (A) mg/ml or HBSS (ontrol) () for 3 min at 35 C. The number of viable bateria in tubes laking PMNs (bateria ontrol) was also measured ( x ). These ounts were obtained by diluting the PMN-baterial suspension in distilled water. The ounts represent surviving intra- and extra-ellular bateria. 11 ounts/2 s. CL peaked at approximately 1-15 min and then gradually dereased over the remainder of the 45 min period. Arylamide pretreatment led to dose-dependent inhibition of CL (Fig. 4). PMNs pre-inubated with arylamide at a onentration of 1 mg/ml had peak CL values about half those of ontrol PMNs, whereas PMNs pre-inubated with arylamide at onentrations of 1- and 1 mg/ml had peak CL values approximately 8% of that of the ontrols. Arylamide-pretreated PMNs showed a more rapid derease in CL during the last 25 min of the 45 min period ompared with the ontrols. Disussion The results presented in this report indiate that arylamide in high onentrations has an inhibitory "o6 K CN -'QD - 4, P 2 E u I 3M Minutes after ativation Fig. 4 Chemiluminesene from ativated PMNs pretreated with various onentrations ofarylamide. The ordinate represents the hemiluminesene emittedfrom PMNs pretreated with arylamide in onentrations of.1 (A), 1k (), and 1 (A) mg/ml or HESS (ontrol) () for 3 min at 35 C. PMNs were ativated by addition of opsonised Staphyloous epidermidis. effet on several human PMN phagoyti funtions in vitro. The ation of arylamide does not appear to be direted at any partiular aspet of the ell biology, suh as movement, respiration or phagoytosis. Whether arylamide bloks eah funtion individually or inativates mehanisms ommon to several or all the funtions is not known. High onentrations (37 5 mg/ml) of arylamide did not affet viability measured by lak of trypan blue uptake. However, hanges in staining properties are insensitive indiators of ell damage and phagoyti integlity, ompared with measurements of respiration and bateriidal apaity (Dankberg and Persidsky, 1976). Arylamide is known to reat with proteins (Drukerey et al., 1953; Hashimoto and Aldridge, 197). Inhibition of phagoyti funtions ould result from arylamide binding to, and inativating, several key enzymes involved in important neutrophil biohemial reations, suh as glyolysis,

6 31 oxygen metabolism, and membrane transport of ations (Karnovsky, 1962; Naahe et al., 1977). Ward and Beker (1967, 1968, 197) and Beker and Ward (1967) have shown that reognition of neutrophil hemotati fators may be dependent upon serine esterases on the ell surfae. Inativation of these proteins by arylamide ould therefore blok their role in phagoyti reognition. Colhiine, whih bloks mirotubule formation, inhibits several PMN funtions inluding hemotaxis, adhesiveness, random motility, degranulation, ingestion, and metaboli stimulation (Bodel and Malawista, 1967; Caner, 1965). In a similar manner, disruption of the polymerisation of tubulin subunits *or an alteration of the mirotubula funtions by arylamide binding to these proteins ould inhibit phagoyti ativity. Drukerey et al. (1953) showed that mixtures of arylamide and protein brines or human serum solidify into a lear gel. Pretreatment of PMNs with arylamide may ause onjugation of proteins on the ell membrane or within the ytosol, thereby dereasing the fluidity of the ell. Alternatively, arylamide bound to the surfae of the phagoyte might ause the serum in the reation mixture to solidify as a gel around eah ell. Dereases in ell flexibility ould explain the diminished random motility, hemotaxis, and bateria (partile) uptake, and would also affet degranulation and metaboli stimulation, whih are dependent on membrane perturbation (Goldstein et al., 1976, 1977). Arylamide is known to polymerise on the surfae of tumour ells beause of the presene of free radials (Buriakova et al., 1966). Prodution of free radials by the PMNs during inubation with arylamide (Allen et al., 1972, 1974) may polymerise arylamide on the PMN surfae, leading to dereased ell flexibility and masking of surfae reeptors. PMN hemiluminesene, a measure of ellular oxidative metabolism, arises from the prodution and subsequent relaxation of high-energy ompounds assoiated with oxygen onsumption during phagoytosis (Allen et al., 1972, 1974; Cheson et al., 1976). As CL orrelates with bateriidal ativity in the PMN (Allen et al., 1972), the derease in killing apaity by arylamide-pretreated PMNs may be attributable to inativation of the bateriidal mehanisms measured by CL. The stimulation of the bateriidal apaities of these ells with onentrations of arylamide below 5- mg/ml was unexpeted beause arylamide in the same onentrations slightly depressed hemotaxis and hemiluminesene. As both movement and oxidative metabolism appear to be inhibited, low D. Mangan and L S. Snyder onentrations of arylamide may enhane other steps of phagoytosis suh as opsonisation (reognition), ingestion, degranulation, or non-oxidative bateriidal systems. The mehanism for this enhanement remains to be investigated. Irrespetive of the in vitro effets, it is unlikely that the aumulation of arylamide would affet PMN funtion in vivo. First, the dose of arylamide neessary to inhibit PMN funtion in vitro (1 mg/ml) appears to be high. It should be stressed that the amount of arylamide whih interats with eah PMN is unknown, as the ells were washed before being tested. The onentration of arylamide that inhibits PMN funtion in experimental animals has not been determined. Seond, arylamide does not attah seletively to PMNs in vivo. Hashimoto and Aldridge (197) found that, after aute intoxiation of test animals, radiolabelled arylamide was bound to protein in many tissues. Whether bound arylamide is not available or is non-toxi for PMNs is unertain, but it seems likely that only those PMNs in the viinity of penetration (and highest onentration) of arylamide would be affeted. Third, PMNs have a half-life of 6 h in the blood, and survive in the body tissues for less than two days (Leeson and Leeson, 197). Thus, the time during whih arylamide an aumulate in a single PMN is limited by the ell's short life span. However, this does not rule out the possibility of inhibitory effets resulting from a single massive exposure to arylamide. The data reported here suggest that additional studies are neessary to determine the effet of arylamide on host suseptibility to infetious agents and on those systems whih determine suh suseptibility. Beause arylamide is used in industrial proesses, its effets must be learly evaluated. Further studies on the mehanism by whih arylamide inhibits phagoytosis are also warranted. This investigation was supported in part by a grant from the West Virginia University Shool of Mediine (BRSG and Medial Corporation Funds). We thank Dr Robert Burrell for his advie in the preparation of the manusript. The seretarial assistane of Mrs Elizabeth Roovih, Mrs Teresa Grimm, and Mrs Lillian Sanga is gratefully aknowledged. Referenes Allen, R. C., Stjernholm, R. L., and Steele, R. H. (1972). Evidene for the generation of an eletroni exitation state(s) in human polymorphonulear leukoytes and its partiipation in bateriidal ativity. Biohemial and Biophysial Researh Communiations, 47,

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