Br. J. exp. Path. (1979) 60, 662
A STUDY OF THE EFFECT OF POVIDONE-IODINE ON POLYMORPHONUCLEAR LEUCOCYTE CHEMOTAXIS J. C. CONNOLLY AND 0. J. A. GILMORE From the Surgical Plrofessorial Unit, St Bartholomew's Hospital, West Smithfield, London, E.C.1 Received for publication June 27, 1979
Summary.-Studies were made of the effect of povidone-iodine on polymorphonuclear leucocyte chemotaxis. Polymorphonuclear leucocytes were incubated with various concentrations of povidone-iodine and allowed to migrate across a membrane towards a chemotactic agent. Chemotactic movement was found to decrease as the concentration of povidone-iodine rose, 75 ,ug/ml completely inhibiting all movement. A concentration of 10 ,ug/ml of povidone -iodine was found actively to repel the white cells. In vivo studies in mice showed a reduction in polymorphonuclear leucocytes at wound surfaces in the presence of povidone-iodine dry powder spray. POLYMORPHONUCLEAR LEUCOCYTE CHEMOTAXIS iS the ability of the cells to react to and move towards a chemical stimulus produced in damaged or infected tissues. These neutrophils are attracted to the injured site by a series of effector substances (Ward, Lepow and Newman, 1968; Ward, Cohen and Flanagen, 1972; Keller and Sorkin, 1967) the mechanism of which is not yet fully understood. Neutrophil migration constitutes the body's first line of defence in the complex inflammatory reaction which is necessary for the eradication of invading organisms. It is therefore of considerable importance if adversely affected. Antiseptics are now being used increasingly in burns units and for the treatment of leg ulcers (Wilkins and Connolly, 1979). Recently there have been many reports of the inhibition by antibiotics of the leucocyte chemotactic response (Forsgren and Schmeling, 1977; Martin et al., 1974; Goodhart, 1977; Majeski and Alexander, 1977) and therefore it is of interest to determine what effect topical antiseptics have upon polymorph chemotaxis. Povidone-iodine in the form of a drypowder spray is employed extensively as an antiseptic agent following surgery. When sprayed into the wound just before
closure it has been shown to reduce infection significantly (Gilmore and Martin, 1974; Gilmore and Sanderson, 1975) without producing any adverse effects on the process of wound healing (Gilmore, Reid and Strokon, 1977). Boyden (1962) devised a simple chemotactic chamber consisting of two compartments divided off by a membrane of such a pore size that leucocytes can only pass through by active migration towards a chemotactic agent placed in the lower compartment. The first experiment reported was undertaken to determine whether the presence of povidone-iodine, in various concentrations relevant to clinical use, would impair the chemotactic activity of polymorphs in such chambers. A further study was undertaken in mice to investigate the effect of povidone-iodine dry-powder spray on wound surfaces. Skin sections were examined histologically. MATERIALS AND METHODS P'reparation of leucocytes. The method of Warden, Mason and Pruitt (1975) was used: 10 ml of heparinized blood was collected from healthy volunteers and diluted 2:1 with a solution of 8%o glucose and 3,333 u/100 ml of heparin in physiological saline. Erythrocyte-mononuclear sedinentation xas carried out in a 50ml conical
POVIDONE-IODINE AND POLYMORPHONUCLEAR LEUCOCYTE CHEMOTAXIS
tube at 370 for 45 min. The leucocyte-rich supernatant, containing approximately 107 viable cells per 10 ml of blood, was removed with a Pasteur pipette. The cells were then diluted 1: 2: 1 (1 vol. cell suspension to 2 vol. Hanks' solution, to 1 vol. phosphate buffer, pH 7-4 (containing povidone-iodine when applicable) respectively). The cells were used within 90 min of the blood being taken. Preparation of chemotactic agent. An overnight pure culture of Escherichia coli 0111 in nutrient broth was centrifuged at 12,000 rpm for 10 min to precipitate the cells. The supernatant was then sterilized by filtration through a Millipore filter of pore size 0-22 ,um and used as the chemotactic agent. Preparation of antiseptic. For use in the upper chamber, povidone-iodine (stored as a 10% solution containing 1% available iodine) was diluted in phosphate buffer, pH 7-4, to the required concentrations. When included in the lower compartment of the blind-well chambers, dilutions were made in Esch. coli culture filtrate, the chemotactic agent. Neutrophil chemotaxis was evaluated in Nuclepore Blind Well chemotactic chambers, simplified copies of Boyden's chamber. 0-2 ml of Esch. coli culture filtrate, pre-warmed to 370, were introduced into the lower compartment. A Nuclepore membrane (13 mm in diameter and with a pore size of 5 tum) was then carefully placed, with the dull side uppermost, above the meniscus. The top of the blind-well chamber was then screwed into place and 0-8 ml of leucocyte preparation was layered on to the upper surface of the membrane. The apparatus was incubated for 2 h at 37°. The effect of povidone-iodine on leucocytes was assessed by the addition of various concentrations of this antiseptic to the white blood cells in the top chamber. The blind-well chambers were again incubated for 2 h at 370. A variety of concentrations of povidone-iodine were then introduced into both chambers to assess the effect of povidone-iodine on chemotaxis throughout the apparatus. After incubation the membranes were carefully removed and fixed with methanol. They were then stained with a May-Grunwald/ Giemsa stain and rinsed in Wright's buffer, pH 6-8, before mounting. The cells were counted the next day. Mouse study. Twenty female Theiller Original mice weighing 20-25 g were anaesthetized with Imobilon. A 1 cm midline incision was made through the abdominal wall to the peritoneal cavity in each mouse. Each incision was then closed in 1 layer with continuous 5/0 Dexon sutures. The mice were randomly assigned to one of two groups. Those in the control group received no treatment, while the remainder had their
663
wounds thoroughly sprayed for 4 sec with povidone-iodine dry-powder spray before and after suturing. Two mice from each group were killed at 1, 21, 5, 8, and 24 h after surgery. The area of incision was excised and sections were examined histologically under high and low magnification. Twenty fields covering the entire length of the incision in each mouse were examined under high power (x 800) and polymorphonuclear leucocyte counts per field were recorded. RESULTS
Chemotaxis was estimated after the introduction of various dilutions of povidone-iodine into the top or both compartments of the blind-well chambers with the leucocyte preparations. Twenty fields for each membrane were examined under the microscope (x 800). The numbers of polymorphonuclear leucocytes on either side of the membrane were counted. Results are expressed as the "chemotactic index" (CI), or "comparative chemotactic index" (CCI), the latter being an expression of the results of the test chamber in comparison with controls (Table I). No. of cells on lower side of membrane x 100% CI -N of No. cells on both upper and lower side of membrane Resulting CI test from chamber x 100% CCICI from control chamber (without antiseptic) The results, expressed as a percentage of that in the control chambers (CCI), show that chemotaxis is inhibited by increasing concentrations of povidoneiodine (Fig. 1). Povidone-iodine at a concentration of 75 ,ug/ml completely inhibits leucocyte chemotaxis. No white blood cells were seen at the lower side of the membrane after incubation for 2 h in the presence of a chemotactic agent and povidone-iodine at this concentration. In the control chambers, where saline replaced the chemotactic agent, there was again no passage of polymorphonuclear leucocytes across the membrane, indicat-
J. C. CONNOLLY AND 0. J. A. GILMORE
664
TABLE I.-Chemotactic Index and Comparative Chemotactic Index at Various Concentrations of Povidone-Iodine Group (a)
(b)
(c)
(d) (e)
Contents of lower chamber Esch. coli
Contents of top chamber Polymorphs P.I. at 10 jug/ml Polymorphs P.I. lO1,g/ml Polymorphs No antiseptic Polymorphs P.I. 10 jug/ml Polymorphs Control
Comparative Chemotactic chemotactic index index > 100°% 100%
Esch. colt P.I. neg/ml 10 Saline
51-8%
85-2%
00,/ 0/O
0%
Saline
305%
50-2%
Esch. coli
60.8%
100%
Where P.I. denotes povidone-iodine.
CCI
*
100%
top) clamber orily C Povi(done-iodline present
Povi(done-iodine
both chambers
80%
present irn
irn
[kg/ml of povidone-iodine tactic index of 51.8%.
gave a
chemo-
Mou.se study
The results of this in vivo study are recorded in Table II. Slides of sections taken through the wound surface, stained 40% with haematoxylin and eosin, were examined histologically. The polymorphonu20% clear leucocyte response to infection was to be at a maximum between 5 and seen 11111 0% , 8 h after surgery. Significantly fewer 0 20 60 70 50 30 40 leucocytes were seen in the areas in the Antiseptic vicinity of wounds sprayed with immediate FIG. 1. Increasing concentrations of povithan in those which had povidone-iodine done-iodine inhibit chemotaxis. Results received no antiseptic treatment. expressed as a percentage of control chambers (C.C.L.). x 000. Of special interest is a wound infection in one mouse from the povidone-iodine ing that random motion through the treatment group, which had been killed membrane was not responsible for the 24 h postoperatively. This slide (Fig. 2) results. When povidone-iodine at a concentra- TABLE II.-Neutrophils in Mouse Tissue at tion of 10 pg/ml was added to the cells in Various Times after Tissue Sprayed with the top chamber, chemotaxis appeared to Povidone-Iodine be considerably enhanced when compared Hours following with concurrently run controls containing Povidonesuturing no antiseptic. On each occasion 100% of iodine Significance until time Control p group group of death the leucocytes migrated through the mem11-0
A STUDY OF THE EFFECT OF POVIDONE-IODINE ON POLYMORPHONUCLEAR LEUCOCYTE CHEMOTAXIS J. C. CONNOLLY AND 0. J. A. GILMORE From the Surgical Plrofessorial Unit, St Bartholomew's Hospital, West Smithfield, London, E.C.1 Received for publication June 27, 1979
Summary.-Studies were made of the effect of povidone-iodine on polymorphonuclear leucocyte chemotaxis. Polymorphonuclear leucocytes were incubated with various concentrations of povidone-iodine and allowed to migrate across a membrane towards a chemotactic agent. Chemotactic movement was found to decrease as the concentration of povidone-iodine rose, 75 ,ug/ml completely inhibiting all movement. A concentration of 10 ,ug/ml of povidone -iodine was found actively to repel the white cells. In vivo studies in mice showed a reduction in polymorphonuclear leucocytes at wound surfaces in the presence of povidone-iodine dry powder spray. POLYMORPHONUCLEAR LEUCOCYTE CHEMOTAXIS iS the ability of the cells to react to and move towards a chemical stimulus produced in damaged or infected tissues. These neutrophils are attracted to the injured site by a series of effector substances (Ward, Lepow and Newman, 1968; Ward, Cohen and Flanagen, 1972; Keller and Sorkin, 1967) the mechanism of which is not yet fully understood. Neutrophil migration constitutes the body's first line of defence in the complex inflammatory reaction which is necessary for the eradication of invading organisms. It is therefore of considerable importance if adversely affected. Antiseptics are now being used increasingly in burns units and for the treatment of leg ulcers (Wilkins and Connolly, 1979). Recently there have been many reports of the inhibition by antibiotics of the leucocyte chemotactic response (Forsgren and Schmeling, 1977; Martin et al., 1974; Goodhart, 1977; Majeski and Alexander, 1977) and therefore it is of interest to determine what effect topical antiseptics have upon polymorph chemotaxis. Povidone-iodine in the form of a drypowder spray is employed extensively as an antiseptic agent following surgery. When sprayed into the wound just before
closure it has been shown to reduce infection significantly (Gilmore and Martin, 1974; Gilmore and Sanderson, 1975) without producing any adverse effects on the process of wound healing (Gilmore, Reid and Strokon, 1977). Boyden (1962) devised a simple chemotactic chamber consisting of two compartments divided off by a membrane of such a pore size that leucocytes can only pass through by active migration towards a chemotactic agent placed in the lower compartment. The first experiment reported was undertaken to determine whether the presence of povidone-iodine, in various concentrations relevant to clinical use, would impair the chemotactic activity of polymorphs in such chambers. A further study was undertaken in mice to investigate the effect of povidone-iodine dry-powder spray on wound surfaces. Skin sections were examined histologically. MATERIALS AND METHODS P'reparation of leucocytes. The method of Warden, Mason and Pruitt (1975) was used: 10 ml of heparinized blood was collected from healthy volunteers and diluted 2:1 with a solution of 8%o glucose and 3,333 u/100 ml of heparin in physiological saline. Erythrocyte-mononuclear sedinentation xas carried out in a 50ml conical
POVIDONE-IODINE AND POLYMORPHONUCLEAR LEUCOCYTE CHEMOTAXIS
tube at 370 for 45 min. The leucocyte-rich supernatant, containing approximately 107 viable cells per 10 ml of blood, was removed with a Pasteur pipette. The cells were then diluted 1: 2: 1 (1 vol. cell suspension to 2 vol. Hanks' solution, to 1 vol. phosphate buffer, pH 7-4 (containing povidone-iodine when applicable) respectively). The cells were used within 90 min of the blood being taken. Preparation of chemotactic agent. An overnight pure culture of Escherichia coli 0111 in nutrient broth was centrifuged at 12,000 rpm for 10 min to precipitate the cells. The supernatant was then sterilized by filtration through a Millipore filter of pore size 0-22 ,um and used as the chemotactic agent. Preparation of antiseptic. For use in the upper chamber, povidone-iodine (stored as a 10% solution containing 1% available iodine) was diluted in phosphate buffer, pH 7-4, to the required concentrations. When included in the lower compartment of the blind-well chambers, dilutions were made in Esch. coli culture filtrate, the chemotactic agent. Neutrophil chemotaxis was evaluated in Nuclepore Blind Well chemotactic chambers, simplified copies of Boyden's chamber. 0-2 ml of Esch. coli culture filtrate, pre-warmed to 370, were introduced into the lower compartment. A Nuclepore membrane (13 mm in diameter and with a pore size of 5 tum) was then carefully placed, with the dull side uppermost, above the meniscus. The top of the blind-well chamber was then screwed into place and 0-8 ml of leucocyte preparation was layered on to the upper surface of the membrane. The apparatus was incubated for 2 h at 37°. The effect of povidone-iodine on leucocytes was assessed by the addition of various concentrations of this antiseptic to the white blood cells in the top chamber. The blind-well chambers were again incubated for 2 h at 370. A variety of concentrations of povidone-iodine were then introduced into both chambers to assess the effect of povidone-iodine on chemotaxis throughout the apparatus. After incubation the membranes were carefully removed and fixed with methanol. They were then stained with a May-Grunwald/ Giemsa stain and rinsed in Wright's buffer, pH 6-8, before mounting. The cells were counted the next day. Mouse study. Twenty female Theiller Original mice weighing 20-25 g were anaesthetized with Imobilon. A 1 cm midline incision was made through the abdominal wall to the peritoneal cavity in each mouse. Each incision was then closed in 1 layer with continuous 5/0 Dexon sutures. The mice were randomly assigned to one of two groups. Those in the control group received no treatment, while the remainder had their
663
wounds thoroughly sprayed for 4 sec with povidone-iodine dry-powder spray before and after suturing. Two mice from each group were killed at 1, 21, 5, 8, and 24 h after surgery. The area of incision was excised and sections were examined histologically under high and low magnification. Twenty fields covering the entire length of the incision in each mouse were examined under high power (x 800) and polymorphonuclear leucocyte counts per field were recorded. RESULTS
Chemotaxis was estimated after the introduction of various dilutions of povidone-iodine into the top or both compartments of the blind-well chambers with the leucocyte preparations. Twenty fields for each membrane were examined under the microscope (x 800). The numbers of polymorphonuclear leucocytes on either side of the membrane were counted. Results are expressed as the "chemotactic index" (CI), or "comparative chemotactic index" (CCI), the latter being an expression of the results of the test chamber in comparison with controls (Table I). No. of cells on lower side of membrane x 100% CI -N of No. cells on both upper and lower side of membrane Resulting CI test from chamber x 100% CCICI from control chamber (without antiseptic) The results, expressed as a percentage of that in the control chambers (CCI), show that chemotaxis is inhibited by increasing concentrations of povidoneiodine (Fig. 1). Povidone-iodine at a concentration of 75 ,ug/ml completely inhibits leucocyte chemotaxis. No white blood cells were seen at the lower side of the membrane after incubation for 2 h in the presence of a chemotactic agent and povidone-iodine at this concentration. In the control chambers, where saline replaced the chemotactic agent, there was again no passage of polymorphonuclear leucocytes across the membrane, indicat-
J. C. CONNOLLY AND 0. J. A. GILMORE
664
TABLE I.-Chemotactic Index and Comparative Chemotactic Index at Various Concentrations of Povidone-Iodine Group (a)
(b)
(c)
(d) (e)
Contents of lower chamber Esch. coli
Contents of top chamber Polymorphs P.I. at 10 jug/ml Polymorphs P.I. lO1,g/ml Polymorphs No antiseptic Polymorphs P.I. 10 jug/ml Polymorphs Control
Comparative Chemotactic chemotactic index index > 100°% 100%
Esch. colt P.I. neg/ml 10 Saline
51-8%
85-2%
00,/ 0/O
0%
Saline
305%
50-2%
Esch. coli
60.8%
100%
Where P.I. denotes povidone-iodine.
CCI
*
100%
top) clamber orily C Povi(done-iodline present
Povi(done-iodine
both chambers
80%
present irn
irn
[kg/ml of povidone-iodine tactic index of 51.8%.
gave a
chemo-
Mou.se study
The results of this in vivo study are recorded in Table II. Slides of sections taken through the wound surface, stained 40% with haematoxylin and eosin, were examined histologically. The polymorphonu20% clear leucocyte response to infection was to be at a maximum between 5 and seen 11111 0% , 8 h after surgery. Significantly fewer 0 20 60 70 50 30 40 leucocytes were seen in the areas in the Antiseptic vicinity of wounds sprayed with immediate FIG. 1. Increasing concentrations of povithan in those which had povidone-iodine done-iodine inhibit chemotaxis. Results received no antiseptic treatment. expressed as a percentage of control chambers (C.C.L.). x 000. Of special interest is a wound infection in one mouse from the povidone-iodine ing that random motion through the treatment group, which had been killed membrane was not responsible for the 24 h postoperatively. This slide (Fig. 2) results. When povidone-iodine at a concentra- TABLE II.-Neutrophils in Mouse Tissue at tion of 10 pg/ml was added to the cells in Various Times after Tissue Sprayed with the top chamber, chemotaxis appeared to Povidone-Iodine be considerably enhanced when compared Hours following with concurrently run controls containing Povidonesuturing no antiseptic. On each occasion 100% of iodine Significance until time Control p group group of death the leucocytes migrated through the mem11-0
