ANTimconRo L AGZNTS AND CHzMOTHERAPY, Oct. 1977, p. 540-542 Copyright © 1977 American Society for Microbiology

Vol. 12, No. 4 Printed in U.S.A.

Effect of Aminoglycosides on the Chemotactic Response of Human Polymorphonuclear Leukocytes GLENN L. GOODHART Philadelphia Veterans Administration Hospital, Medical College ofPennsylvania Infectious Diseases Service, Philadelphia, Pennsylvania 19104 Received for publication 29 June 1977

Leukocyte chemotaxis was inhibited 9.6% compared with control in the presence of 10 ,ug of gentamicin per ml and 10.5% when exposed to 20 ,g of amikacin per ml. However, cells incubated with an injectable form of gentamicin, containing preservatives, were inhibited an additional 25.8% relative to cells incubated with pure gentamicin.

Gentamicin has recently been reported. to strongly inhibit the chemotactic response of human polymorphonuclear leukocytes (PMNs) (A. J. Kahn, H. E. Evans, L. Glass, and P. Khan, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 15th, Washington, D. C., Abstr. no. 65, 1975; Khan et al., Clin. Res. 23:587, 1975; and Khan et al., Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 16th, Chicago, Ill., Abstr. no. 108, 1976). However, Majeski et al. (3, 4) found only minimal inhibition of chemotaxis even when with high concentrations of gentamicin. The present report is an attempt to resolve this inconguity and to extend the results to amikacin-the newest available aminoglycoside. PMNs were prepared by dextran sedimentation and hypotonic lysis of heparinized whole venous blood (2) from 14 healthy donors. PMNs were counted by hemocytometer and suspended in Hanks balance salt solution with 2% bovine serum albumin (HBSS-BSA). The chemotactic factor (CF) was casein (5 mg of casein per ml of HBSS-BSA). Triplicate modified Boyden chambers were used. A 3-,um micropore filter (Millipore, Bedford, Mass.) separated the top and bottom compartments of each chamber. The bottom compartments of control chambers were filled with HBSS-BSA (unstimulated) or CF (stimulated), and then 6 x 105 PMNs were placed in the top compartments. Experimental chambers were similarly prepared except that both compartments of each chamber contained either 10 ,g of gentamicin or 20 ,ug of amikacin per ml. The chambers were placed in a humidified incubator for 90 min at 37C. The filters were immediately removed, fixed, stained, and mounted (5). The distance from the monolayer to a level where 2 to 8 PMNs per high-powered field (hpf) could be counted was determined in a single filter from an unstimulated control

chamber with the micrometer on the fine focus adjustment of the microscope. The PMNs per hpf of all filters from an experiment were then determined at this standard depth (1). Five fields from each filter were counted, and the arithmetic mean of the triplicate chambers was calculated. To assess whether aminoglycosides influenced random motion of PMNs, PMNs from five subjects were isolated. The top and bottom compartments of duplicate experimental chambers were filled with 10 ,ug/ml, final concentration, of gentamicin. In contrast to the previous experiments, the top and bottom compartments of each chamber contained equal final concentrations of CF. A comparison was made between injectable and powdered forms of gentamicin. Injectable gentamicin is sold in 1-ml vials containing 40 mg of gentamicin, 1.8 mg of methylparaben, and 0.2 mg of propylparaben. PMNs from four subjects were preincubated for 10 min at 370C with either 10 ,ug of the powder form (preservative-free) of gentamicin or the injectable form (containing preservatives) per ml and then studied in a CF concentration gradient containing antibiotics as outlined above. Because of the method of determining the PMNs per hpf in the unstimulated control chambers, the mathematical requirements for t-test comparison to the unstimulated experimental chambers were not met. However, the comparison of gentamicin-treated to control cells response to a concentration gradient of CF is statistically valid and is shown in Table 1. Gentamicin very slightly but significantly decreased the PMNs per hpf in stimulated chambers (P < 0.01, t test). Whereas the effect of gentamicin on stimulated PMNs was significantly different than control, the inhibition was only 9.6% compared with that of control. Amikacin also decreased (Table 1) the PMNs per



VOL. 12, 1977

hpf of stimulated chambers (P < 0.005). Again, the inhibition was only 10.5% compared with control. The effect of gentamicin on the random migration of PMNs is shown in Table 2. The response of control PMNs to increasing nongradient concentrations of CF was biphasic: there was an increase in random motion until a point where higher concentrations of CF became inhibitory (6). No differences (P > 0.05) were found between gentamicin-treated or control cells. It therefore appears that the slight inhibition by aminoglycosides of PMNs exposed to concentration gradients of CF is the result of impaired, directed migration. A comparison was made between the effect of injectable and preservative-free gentamicin (Table 3). In four subjects, chemotaxis of PMNs incubated with injectable gentamicin was inhibited 25.8 + 10.3% in addition to the impairment observed in PMNs incubated with preservative-free gentamicin. The studies of Majeski et al. found no inhibition of chemotaxis by 10 ,ug/ml but slight inhibition with higher concentrations of gentami-


cin, which is compatible with the present study. The reports of Khan et al. (15th ICAAC, Abstr no. 65, 1975. Clin. Res. 23: 587, 1975; 16th ICAAC, Abstr. no. 108, 1976), however, are not. They reported a depressed response to a concentration gradient of chemotactic factor (endotoxin-activated serum) of PMNs obtained from volunteers given gentamicin. Also, in in vitro experiments PMNs preincubated with gentamicin were inhibited about 40% of control. A technical problem was studied to determine if it could explain the differences between the Khan et al. studies and the present one. As noted above, injectable gentamicin contains preservatives that may have influenced the chemotaxis assay independently of the antibiotic. As shown in Table 3, cells exposed to injectable gentamicin did not respond as well to CF as cells exposed to powdered gentamicin (injectable form inhibited 25.8% more than powdered). Because the Khan et al. reports are in abstract form, further comparison of data would be speculative. In conclusion, neither gentamicin nor amikacin caused major impairment of the chemotac-

TABLE 1. Effect of aminoglycosides on response of PMNs to concentration gradient of CF Aminoglycosides Bottom compartment of Boyden chamber contained (,g/ml);

Top compartment of Boyden chamber contained



HBSS-BSA HBSS-BSA HBSS-BSA CF HBSS-BSA, HBSS-BSA, gentamicin (10) HBSS-BSA, CF, gentamicin (10) HBSS-BSA, HBSS-BSA, amikacin (20) HBSS-BSA, CF, amikacin (20) a n = 14; results in mean ± standard deviation.

5.3 127.6 16.4 116.4 17.8 115.5

gentamicin (10) gentamicin (10) amikacin (20) amikacin (20)

+ + + ± ± ±

0.6 11.6 4.2 9.4 5.9 8.4

TABiL 2. Effect of 10 pg of gentamicin per ml on response ofPMNs to nongradient concentrations of CF PMNs per hpf







34.7 ± 24.2 24.1 ± 7.7 10.3 ± 11.1 11.3 ± 7.7 10.0 ± 7.1 10.2 ± 9.0 36.5 ± 28.2 21.2 ± 10.5 12.3 ± 7.9 8.2 ± 7.5 a Casein in top and bottom compartment (milligrams per milliliter). n = 5; results in mean ± standard deviation. 10 0

TABLz 3. Comparison ofeffect ofpowdered gentamicin (genta) to injectable gentamicin (genta plus pres) on chemotactic response Aminoglycoside Bottom compartment of Top compartment of Boyden chamber conBoydn chmbercontined chamber Boyden tamed (g/ml): (ILgIml):containedtand(gm) HBSS-BSA, genta (10) CF, genta (10) HBSS-BSA, genta + pres (10) CF, genta + pres (10) a n = 4; results in mean ± standard deviation.

[(1 - PMNs per hpfgenta + pres/PMNs per hpf genta)] x 100%a

25.8 ± 10.3



response of human PMNs. The present study supports the work of Majeski et al. (3, 4). Injectable gentamicin (containing preservatives) did cause an impaired chemotactic response compared with gentamicin powder and probably explains the conflicting reports of Khan et al.


Pauline Gashi Myers provided technical assistance. Gerald L. Mandell kindly reviewed the manuscrpt. Funds were provided by an institutional grant from the Veterans Administration. This paper was presented at the 10th International Congress of Chemotherapy, Zurich, Switzerland, 18-23 September 1977.

ANTIMICROB. AGENTS CHEMOTHER. LITERATURE CITED 1. Goetzl, E. J., and K. F. Austen. 1976. Structural determinants of the eosinophil chemotactic activity of the

acidic tetrapeptides of eosinophil chemotactic factor of anaphylaxis. J. Exp. Med. 144:1424-1437. 2. Harris, M. B., I. Djerassi, E. Schwartz, and R. K. Root. 1974. Polymorphonuclear leukocytes prepared

by continuous-flow filtration leukapheresis: viability

anf function. Blood 44:707-713. 3. Majeski, J. A., M. A. McClellan, and J. W. Alexander. 1975. Evaluation of leukocyte chemotactic response in the presence of antibiotics. Surg. Forum 26:83-85. 4. Majeski, J. A., M. A. McClellan, and J. W. Alexander. 1976. Effect of antibiotics on the in vitro neutrophil chemotactic response. Am. Surg. 42:785-788. 5. Wilkinson, P. C. 1974. Chemotaxis and inflammation, p. 171. Churchill Livingstone, Edinburgh. 6. Wilkinson, P. C. 1974. Chemotaxis and inflammation, p. 35-40. Churchill Livingston, Edinburgh.

Effect of aminoglycosides on the chemotactic response of human polymorphonuclear leukocytes.

ANTimconRo L AGZNTS AND CHzMOTHERAPY, Oct. 1977, p. 540-542 Copyright © 1977 American Society for Microbiology Vol. 12, No. 4 Printed in U.S.A. Effe...
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