Neutrophil Chemotaxis by Propionibacterium acnes Lipase

Transcription

1 INFECTION AND IMMUNITY, Jan. 1982, p /82/ $02.0O/0 Vol. 35, No. 1 Neutrophil Chemotaxis by Propionibacterium acnes Lipase and Its Inhibition WEI-LI LEE,'* ALAN R. SHALITA,1 KAMALA SUNTHARALINGAM,2 AND SENIH M. FIKRIG2 Department of Dermatology' and Department of Pediatrics,2 State University of New York, Downstate Medical Center, Brooklyn, New York Received 20 February 1981/Accepted 27 August 1981 The chemoattraction of Propionibacterium acnes lipase for neutrophils and the effect of lipase inhibitor and two antibiotic agents on the chemotaxis were evaluated. Of the various fractions tested, partially purified lipase (fraction 2c) was the most active cytotaxin produced by P. acnes. Serum mediators were not required for the generation of chemotaxis by lipase in vitro. Diisopropyl phosphofluoridate at low concentration (10-4 mm) completely inhibited lipase activity as well as polymorphonuclear leukocyte chemotaxis generated by lipase. Tetracycline hydrochloride and erythromycin base at concentrations of 10-1 mm and 1 mm, respectively, caused 100%o inhibition of PMN migration toward lipase or zymosan-activated serum. The inhibiting activity of the antibiotics was directed against cells independently of any effect on lipase. Chemotaxis by P. acnes lipase suggests a wider role for this enzyme in the inflammatory process and the pathogenesis of acne vulgaris. The etiological events which ultimately lead to acne lesions are complex and multifactorial, involving androgenic stimulation of the sebaceous gland with resulting increased sebum production, hydrolysis of sebum triglycerides by the follicular microflora, alterations in follicular epithelial differentiation, and a mixed inflammatory infiltrate. The fact that large numbers of Propionibacterium acnes occur in all stages of the acne process and the successful use of antibacterial therapy in the treatment of acne suggest that microbial components play an important role in the disease. Lipase, the extracellular enzyme derived from P. acnes, is reported to be responsible for the hydrolysis of sebum triglycerides to free fatty acids which have been implicated as both irritants and comedogenic agents and which lead to an intensification of the inflammatory process (8, 12, 22, 23). Recent reports, however, suggest that the role of this organism may be more complicated (13, 24, 31). A macrophage-specific cytotoxin derived from anaerobic diptheroids has been reported by Wilkinson et al. (32, 33). It is possible that chemotaxis may be the mechanism for the initiation of inflammatory reactions after release of bacterial products into the dermis. In contrast to the wealth of the information available on the composition and profile of toxins or allergins of most pathogenic bacteria, the corresponding data on P. acnes are poorly documented. Research in the immunological aspect has also been hampered by a lack of purified antigens. One of the major criticisms of most published studies is that the antigen preparations are usually concentrated culture supernatants and are complex in nature and of unknown composition. It is essential to isolate and analyze well-defined antigens to understand the biological and immunological roles of this organism. This investigation was undertaken to determine whether purified lipase from P. acnes was chemotactic for neutrophils. MATERIALS AND METHODS Cultivation of microorganism. P. acnes was cultured as described previously (14). The organism was activated by repeated subculture in the same medium used for the large scale batch culture. To assure anaerobiosis, the broth was gassed with a mixture of N2-CO2 (95:5, vol/vol) before inoculation. Organisms were grown in a shaking metabolic incubator (100 rpm) at 35 C for 5 to 6 days. The late log phase culture supernatants were obtained by centrifugation at 8,000 x g for 40 min in a Sorvall RB-2 centrifuge. Purification of lipase. The preparation of lipase was carried out by the procedures described by Pablo et al. (17) and Hassing (10), with some modifications. These include the ultrafiltration with an Amicon hollow fiber concentrator (model DC2) excluding substances of molecular weights below 10,000 and precipitation with absolute alcohol to 50%6 saturation, followed by gel filtration on Sephadex G-100, and two ion-exchange chromatographies on DEAE-Sephadex A-50 and CM Bio-Gel columns. The Sephadex G-100 chromatography was performed in distilled water or 20 mm sodium acetate buffer (ph 5.5). The DEAE-Sephadex chromatography was performed by elution with an increasing salt gradient in phosphate buffer. The subsequent CM Bio-Gel chromatography was carried out by using a 71

2 72 LEE ET AL. two-step gradient from 0.8 M NaCl in 20 mm sodium acetate buffer (ph 5.5, 600 ml) to 0.02 M NaCl in the same buffer (400 ml). The eluents were collected in an LKB automatic fraction collector with a UV monitor scanning at 280 nm and assayed for lipase activity. The lipolytic fractions were pooled and either dialyzed exhaustively against distilled water or concentrated and desalted in an Amicon ultrafiltration cell (Diafol 402) equipped with an XM 50 membrane, lyophilized, and stored at -70 C. All enzyme purification procedures were carried out at 4 C. The catalytic activity of the lipase was routinely assayed with a Radiometer ph-stat using an olive oilgum arabic emulsion substrate (20). The specific activity of the lipase preparation was expressed in units per milligram of protein. One unit is equivalent to the production of 1,umol of free fatty acids per min. Protein was determined by the Folin phenol reagent with bovine serum albumin as the standard (15). Polyacrylamide-gel electrophoresis. The purity of lipase was examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10o gel by a modification of the method of Weber and Osborn (28) in which 0.1% sodium dodecyl sulfate was used. Lyophilized samples (0.02 to 0.05 mg) were dissolved in sample solution containing 2-mercaptoethanol incubated at 37 C for 1 h and applied to the gel. Electrophoresis was carried out at 4 ma/tube for 3 to 4 h with bromophenol blue as the marker dye. After the completion of electrophoresis, the gels were first fixed and stained in a solution of 0.2% Coomassie blue-509 methanol-5% alcohol and then destained in 7% acetic acid-5% methanol. Molecular weights were determined by comparison with a series of standard proteins run under the same condition: transferin (84,000), bovine serum albumin (66,500), ovalbumin (45,000), chymotrypsinogen (25,700), myoglobin (16,900), cytochrome c (13,000), and lysozyme (14,400) (28). Neutrophil chemotaxis. Neutrophil chemotaxis was assayed in vitro as previously described (5, 7). Briefly, human neutrophils obtained from postpartum blood were isolated by a mixture of Methocel-isopaque (3), washed and resuspended in TC 199 (GIBCO Laboratories) containing 2% bovine serum albumin at a final concentration of 2.5 x 106 polymorphonuclear leukocytes (PMN)/ml. Samples of the cell suspension were then placed in the upper compartment of a modified Boyden chemotactic chamber separated from a chemotactic or control substance (TC 199) by a 5-pm membrane filter (Millipore Corp.). After incubation for 3 h at 37 C in a humidified atmosphere of 5% C02, the filters were removed and stained, and cell migration was quantified. The chemotactic activity was evaluated by taking the average counts in five random highpower fields (HPF) of the number of PMN found on the lower filter surface. Results of chemotaxis activity were expressed in PMN/HPF after subtracting random migration. Duplicate chambers were used for each material tested. The standard error of the mean for the duplicate filters was less than 5%. The day to day variability between tests sequentially in a single control ranged between 5 to 10%. The ability of P. acnes lipase to generate serumderived chemotactic factors was assayed by incubating various isolated fractions with an equal volume of 20%o serum for 30 min at 37 C before adding to the lower compartment. The chemotactic activity of the INFECT. IMMUN. partially purified lipase and lipase-generated serum was tested in a dose-response fashion by either adding various amounts of enzyme preparation (fraction 2c) in TC 199 and applying directly to the lower chamber or by incubating the enzyme with an equal volume of serum for 30 min at 37 C, and TC 199 was used to dilute the serum to 10%o before use. Zymosan-activated serum (ZAS) at a concentration of 1 mg/ml was used as a reference standard. In the inhibition studies, stock solution (0.1 M) of antibiotics and diisopropyl phosphofluoridate (DFP) were prepared in TC 199 and isopropyl alcohol. Serial dilutions of these compounds were made in TC 199. Lipase and ZAS were treated with an equal volume of antibiotic or DFP to achieve the appropriate concentration for the lower compartment. The mixtures were incubated at 37 C for 30 min, and the residual lipase activity was determined by the standard ph-stat method. To determine the effect of these compounds on chemotaxis, the lipase (0.01 mg/ ml), ZAS (1 mg/ml) containing antibiotic, or DFP was added directly to the lower compartment unless stated otherwise. Similar measurements were made in filters obtained from control chambers containing TC 199 (or TC 199-isopropyl alcohol when DFP was used) with or without the chemoattractants. The percentage of inhibition was calculated by comparison of the chemotactic activity obtained with cells migrating towards chemoattractants and toward antibiotic- or DFP-treated chemoattractants. To study the inhibitory effect of antibiotic directed against cells, neutrophils (5 x 106 cells per ml) were incubated in a shaking water bath at 37 C for 30 min in the presence of an equal volume of 0.02 mm antibiotics. After incubation, the residual chemotactic responsiveness of the treated cell was tested in chemotactic chambers by using the bacterial factors in the lower compartment. RESULTS Purification of lipase. The ethanol precipitates were fractionated on Sephadex G-100 by eluting with water or sodium acetate buffer. In both systems it separated to give a peak (fraction la) in the excluded volume and a broad peak containing small-molecular-weight polymers and salts (Fig. 1). Rechromatography (not shown) on a column of Sephadex 200 eluting with the same buffer gave a single broad peak (a portion of which was in the excluded volume). These results indicate that lipase has a molecular weight in the 150,000 to 200,000 range. The lipolytic activity was found to be confined to the highmolecular-weight peak and was combined and fractionated on a DEAE A-50 column. The lipase was not absorbed by the anionic column and was eluted out before the gradient started. The second cationic chromatographic proffle is shown in Fig. 2. The enzyme assay of the fractions (2a, 2b, and 2c) showed that they varied in lipolytic activity, and the fraction that eluted at 0.55 M NaCl contained greater than 90% of the total lipase activity. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of the purified lipase

3 VOL. 35, 1982 NEUTROPHIL CHEMOTAXIS BY P. ACNES cr r -U (n m T,) C-) C1 FRACTION FIG. 1. Chromatography on Sephadex G 100 of products supernatant. obtained by ethanol precipitation of culture (fraction 2c) is shown in Fig. 3. The major protein band appeared homogeneous migrating at a mobility similar to that of the protein standard bovine serum albumin with an estimated molecular weight of 65,000 ± 3,000. In addition, one minor protein component with moderate mobility of 25,000 was also present. Neutrophil chemotaxis and inhibition. Fractions isolated throughout the lipase purification procedure and zymosan were assayed for neutrophil chemotactic activity (Fig. 4). All fractions except fraction 2a, the low salt eluted CM Bio-Gel fraction, demonstrated chemotactic activity. Fraction 2c was the most active cytotaxin among the fractions. In contrast to zymosan, evi i2 a 2b 2c GRADIENT which required compiement components for its chemotactic activity, the lipase exerted a direct chemotactic effect on PMN in the absence of serum. The amount of chemotactic activity produced was directly proportional to the amount of P. acnes lipase added (Fig. 5). When >15,ug of enzyme was used, chemotactic activity produced by lipase fell rather sharply. The effect of excess enzyme was qualitatively similar to that observed with lipase-generated serum. The presence of serum inhibitor of the P. acnes lipase probably accounts for the larger concentration of enzyme required to generate comparable chemotactic activity in serum. The results of enzyme inhibition by various 0. (A r- -D m --i Ca 0.2 1O FRACTION FIG. 2. Chromatography of P. acnes lipase on CM Bio-Gel. The dashed line shows the concentration of NaCl in the gradient. The fractions were pooled as indicated by the bar lines. 10 0

4 74 LEE ET AL. INFECT. IMMUN. 0 LcrPASE OAO IYMO ---T ,.2 4 :. t, O RELAT VE MOBIL 'y FIG. 3. Sodium dodecyl sulfate (10%) polyacrylamide gel pattern and molecular weight of purified lipase. Arrows indicate the position of protein bands of P. acnes lipase. compounds are shown in Table 1. It is apparent that tetracycline and DFP were effective lipase inhibitors, and the degree of inhibition was concentration dependent. Completely lipolytic inhibition was found at concentrations of 1 mm and 10-4 mm for tetracycline and DFP, respectively. In contrast, erythromycin demonstrated no effect upon the lipase. Similar conclusions were made by Weeks et al. (31), except that the DFP inhibition was found more pronounced with fraction 2c. The effects of these compounds on PMN migration are shown in Fig. 6A and B. LL aī Cf, z a- L w z z wcc L Both antibiotics suppressed the chemotactic activity of ZAS, and linear inhibition was found at concentrations between 0.01 and 0.1 mm tetracycline, and at concentrations between 0.5 to 1 mm erythromycin. The antibiotic inhibition was not specific to the chemotactic agent. Parallel dose-response curves were found when either lipase or ZAS was used as the chemotactic stimulus. It should be noted, however, that the degree of inhibition appears to be greater when lipase is used as the chemoattractant. The effect of DFP on neutrophil chemotaxis Zy la 2a 2b 2c (1mg) (0.1mg) (0.1lmg) (0.01 mg) FIG. 4. Neutrophil chemotaxis. Hatched bars represent mean value for neutrophil chemotaxis ±1 standard deviation when serum is supplemented with bacterial factor. Open bars represent comparable results by adding bacterial factor alone. Values given = PMN migration (TC chemoattractant) - PMN random migration (TC 199).

5 VOL. 35, 1982 NEUTROPHIL CHEMOTAXIS BY P. ACNES 75 Li. a z 0. w co z w P. ACNES LIPASE (,Ig) FIG. 5. Chemotactic activity of neutrophils in response to P. acnes lipase (0) and lipase-generated serum (0). Negative control activity was 3.5. Positive control (ZAS) activity was (Fig. 6B) was found to be quite different from that of the antibiotics. The DFP effect was specific for lipase and showed no effect on ZAS at concentrations ranging from 10-3 to 10-6 mm. The dose-response curve was characterized by a high initial response slope at low concentrations of DFP. Additional information on whether DFP (10-3 mm) is inhibitory to cell-directed or lipasedirected chemotactic activity was obtained by using a Bio-Gel P 2 column which excluded DFP from DFP-treated lipase. No significant migration occurred when the eluting lipase (0.01 mg/ ml) was tested in the lower compartment. These results suggest that the inhibition by antibiotics appears to be independent of their effect on the lipase. Further experiments were conducted to determine whether incubation of PMN with antibiotics could result in a sup (A)~ Z 0/ z50.i TABLE 1. Inhibition of P. acnes lipase activity Compounda Concn (mm) % Inhibition Tetracycline lo Erythromycin 10-' Diisopropyl fluorophosphate a Compounds were incubated at 37 C for 30 min with the enzyme preparation (2c) before the assay for residual lipase activity. pressed chemotactic response. PMN were incubated at 37 C for 30 min in TC 199 containing 0.01 mm antibiotics. Cells suspended in bovine serum albumin containing TC 199 were either washed or directly placed in the Boyden chamber and tested for their chemotactic responses by using the lipase or ZAS as a chemoattractant. Controls consisted of cells incubated in the same manner with TC 199. The chemotactic activity (PMN/HPF) was compared to the results obtained from the chambers containing cells alone. The antibiotic (0.01 mm) was also placed in the lower compartment to assess if chemotactic activity was present. Preincubation with antibiotics caused a 60 and 38% inhibition with tetracycline and erythromycin, respectively, when cells were tested against lipase and an inhibition of 31 and 27%, respectively, when cells were tested against ZAS (Table 2). The concentration of antibiotic used in this experiment was considerably smaller than required to inhibit a similar degree of neutrophil chemotaxis when the antibiotic was incubated with the chemotactic factor. Since the antibiotic alone showed minimal (B) of &-4 g0o INHIBITOR CONCENTRATION (mm) FIG. 6. Inhibitory effect of antibiotics (A) and DFB (B) on neutrophil chemotaxis. Various concentrations of tetracycline (T), erythromycin (E), and DFP were incubated with lipase (2c) or zymosan activated serum (ZAS) as described in the text. Symbols: 0, T + 2c; A, T + ZAS; 0, E + 2c; A, E + ZAS; *, DFP + 2c; x, DFP + ZAS.

6 76 LEE ET AL. INFECT. IMMUN. TABLE 2. Effect of preincubation with antibiotics on subsequent chemotactic activity of human neutrophils Neutrophil chemotaxis Test samplea Chemoattractant NMN/HPF PMN/HPFb cnemoiaion % to Inhibition' Cell lipase 121 Cell + T lipase Cell + E lipase Cell + T then washed lipase Cell + E then washed lipase Cell ZAS 106 Cell + T ZAS Cell + E ZAS Cell + T then washed ZAS Cell + E then washed ZAS a Neutrophils were incubated at 37 C for 30 min with 0.01 mm of antibiotic in TC 199. After incubation, cells were either added directly to the upper compartment of the chemotactic chamber or washed to remove the antibiotic before testing for the chemotactic activity. E, Erythromycin; T, tetracycline. b Results are shown in PMNtHPF after subtracting random migration in controls. c In this study, duplicate experiments had coefficients of variation ranging between 5 or 10%. All values less than 10% inhibition would therefore be considered insignificant. chemotactic activity (5 and 2 PMN/HPF for tetracycline and erythromycin, respectively), the observed suppression in chemotaxis after preincubation was not the result of subjecting PMN to the chemotactic factor. It should be noted that when antibiotic was placed with PMN in the upper compartment throughout the assay, the degree of antibiotic inhibition increased significantly (P < 0.001) when tested against ZAS. DISCUSSION The effects of various bacterial products upon the induction of immune responses and their interaction with leukocytes have been well studied. The anaerobic diphthroid Corynebacterium parvum, which appears to be the same organism as Propionibacterium acnes and Propionibacterium granulosum (4), has excited considerable interest in recent years because it markedly influences the course of immune responses. Thus, it acts as stimulant of the reticuloendothelial system, as an adjuvant to enhance humoral and cell-mediated responses, and as an antitumor agent which modifies the immunological response in tumors (1, 9, 16, 21). We have studied the chemotactic activity of P. acnes lipase which acts directly toward neutrophils in the absence of serum. The characteristics of this factor appeared to be similar to the macrophage-specific chemotactic factor reported by Wilkinson et al. (33). The production of both factors, elaborated at late log phase plateaued at 6 to 8 days before leveling off coincided with lipase production and was independent of the active cell growing log phase (3 days). They both are macromolecules and stable to heat (56 C), indicating that the tertiary structure is probably not involved in its function. The originally reported macrophage-specific chemotactic factor now appears to be active for neutrophils as well (personal communication). The chemotactic activity of lipase toward other cell types requires further examination. In agreement with its eluting behavior on gel filtration, P. acnes lipase and the majority of microbial lipases have been reported to be large molecules of aggregates of protein-lipid or protein-protein. A structure of this type makes it possible to suggest that the heterogeneity observed on CM Bio-Gel is due to the presence of molecules having different portions of the same basic structure. It also explains some of the confficting data on the size and specificity of P. acnes lipase as described by various groups who isolated cells from different growth phases ((H. H. Ku, personal communication) or by various procedures (10, 17). The chemotactic factors described here apparently differ from those that were reported by Keller and Sorkin (11) and Ward et al. (19, 26, 27) in that they are low molecular weight (150 to 1,500 A), their production is related to the active growth phase of bacterial replication, and they are known to act indirectly by activating complement or a similar enzyme system in plasma. Similar chemotactic factors are also produced by P. acnes (18, 29, 30). The study dealing with neutrophil inhibition after treatment with antibiotics or lipase inhibitor is of special interest with regard to the pathogenesis of acne vulgaris and confirms similar observations by Esterly et al. (6). The mechanism by which antibiotics might affect neutrophil mobility is not clear. Evidence at this time suggests that a cellular mode of action is at least

7 VOL. 35, 1982 partially responsible for chemotactic inhibition. This conclusion is supported by the results in Table 1 and Table 2 indicating that although erythromycin does not inactivate the lipolytic activity of lipase, the preincubation of PMN containing the antibiotic results in suppressed chemotactic responsiveness. In addition, this cell-direct inhibition is not chemotactic factor specific as they reduced cell mobility toward lipase as well as toward ZAS. Suppression at the cellular level could explain the observed nonspecific inhibition of chemotactic agents. However, the greater suppressive effect observed when antibiotic was present throughout the assay in the ZAS system may support the presence of both cell-direct and factor-direct inhibitor as the latter was caused after the contact of the cell and ZAS. Diisopropyl phosphofluoridate and other analogs of phosphonate ester have been known to irreversibly inhibit the serine esterase of neutrophils at concentration of 10-5 M or less (2, 25). In the present study, DFP has been shown to exhibit a marked inhibitory effect on neutrophil chemotaxis against bacterial lipase. A correlation seems to exist between the inhibition of catalytic activity and the inhibition of chemotaxis by DFP as the two phenomena are affected by the same concentration. It is possible that serine residue on the active site of P. acnes lipase (17) may also be involved in attracting neutrophils. Chemotaxis may be affected by inhibiting either the chemotactic factor or the migrating response of the cell under study. In this investigation we have demonstrated that under our experimental conditions the inhibition of neutrophil chemotaxis by antibiotics is due to a direct effect on the neutrophil rather than on the chemotactic factor. The relatively low concentrations required for this inhibition suggest that antibiotics may play an additional anti-inflammatory role when used in the treatment of acne. ACKNOWLEDGMENT This research was supported in part by a grant from the Dermatology Foundation. LITERATURE CITED 1. Adlam, C., and M. T. Scott Lymphoreticular stimulatory properties of Corynebacterium parvum and related bacteria. Med. Microbiol. 6: Becker, E. L Biochemical aspects of the polymorphonuclear response to chemical factors, p In R. Austin and E. L. Becker (ed.), Biochemistry of the acute allergic reactions. Blackweli, Oxford. 3. Boyum, A Separation of white blood cells. Nature (London) 204: Cummins, C. S., and J. L. Johnson Corynebacterium parvum: a synonym for Propionibacterium acnes. J. Gen. Microbiol. 80: Edelson, P. J., E. P.Stltes, S. Gold and H. H. Fudenberg. NEUTROPHIL CHEMOTAXIS BY P. ACNES Disorders of neutrophil function: defects in the early stages of the phagocytic process. Clin. Exp. 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