British Journal of Haematology, 1975, 29, 273.
The Effect of Cytotoxic and Anti-inflammatory Drugs on the Phagocytosis, of Neutrophil Leucocytes J. A. WHITTAKER, H. R. HUGHES AND M. KHURSHID Departwent of Haenzatology, W e l s h Natioiial School oJ’M&iizc, C a r d i f (Received 22 April 1974; ncccptedfor pirblicntioii 3 J
i m
1974)
SUMMARY. The phagocytosis of hcat-killcd Candida albicarzs by iicutrophil lcucocytes was inhibited irz vitro by phciiylbutazonc and acctylsalicylic acid and by the cytotoxic drugs viiicristine, vinblastinc, cytosiiic arabinoside, dauiiorubiciii and busulphan, but not by mustiiic liydrochloridc or procarbazinc. In vivo studies in man havc shown a significant reduction in pliagocytosis after plicnylbutazoiic ingestion, but not following an oral dose of aspirin. Possible explanatory meclianisms of drug action on neutrophil phagocytosis arc discusscd. Sevcral chemically unrelated drugs have been sliowii to depress phagocytosis iii vitro. These iiiclude the anti-inflammatory drug phenylbutazonc (Straws ct a/, 1968; Ruutu & Kosunen, 1972) and tlic phciiothiazincs (Ruutu, 1972). A variety of cytotoxic agents is now in use for the treatment of maligiiaiit disease but many of these agents cause iieutropciiia and their usc is associatcd with an increased incidence of infection. In the prcseiit study we havc measured tlic cffcct of scveral cytotoxic agents 011 neutrophil phagocytic function and attempted to cor rclatc tlic known iir vifro depression of yhagocytosis caused by the anti-iiiflanimatory drugs phciiylbutazonc and acetylsalicylic acid with their effect iit vivo in human subjects. MATERIALS A N D METHODS
Drugs. The concentration of drugs uscd for itl vitro cxpcriiiiciits (Tablc I) was cquivalciit to coiiceiitratioiis achieved iii vivo by therapeutic dosage. Fresh solutions of each drug wcrc prepared immcdiatcly bcforc each cxpcriinciit and 0 .I 1111 of thc first drug solution dissolved in distillcd water or otlicr appropriatc solvc~itwas incubated with 0.9 1111 of thc lcucocytc suspciisioii. CoiitroI cultures wcrc prepared excluding tlic drugs but iiicludiiig thc solvent uscd to dissolve thc drug. Mdia arid reagents. (i) Hanks balaiiccd salt solution (HEJSS) without antibiotics. (ii) A heatkillcd suspension of Caridida albicam, 6 x 106 cclls/ml. (iii) EDTA solution 0.9% in sodium cliloridc 0.9%. (iv) Sterile 6”/, dcxtraii (in01 wt 150 000) in 0.9% sodium chloride. Preparation qf lezrcocytc strspcmion. 20 1111 vciious blood samples wcrc collcctcd with plastic syriiigcs from five hacmatologically noriii.al voluiitccrs into plastic bottlcs containing lithium heparin (Staync Laboratories Ltd) a i d 0.5 nil of dcxtraii/salinc solutioii. The red cells were Correspondence: Dr J. A. Whittnkcr, Dcpartnicii t o f Hncinatology, Wcluh Natioiial School of Mcdiciiic, I-lcath Park, Cardiff CF4 4XW.
273
J. A. Whittaker, H. R. Hughes and Ad. Khtdrshid
274
allowed to sediment at room temperature for 30 iiiiii and the leucocyte rich supernatants harvested, pooled and Centrifuged. The leucocytc deposit was washed twice in HBSS and finally resuspeiidcd in HBSS to give a final concciitratioii of leucocytes of 5 x 106 cells/ml. The superiiataiit plasma from the pooled fractioiis was saved and used to resuspend the leucocytes after incubation with thc drugs. Measrircnieiit of the ykugocytic indcx. The pliagocytosis of Curidida albicuns particles was measured by a modification of thc method of Millcr & Nilsson (1970). Equal volumes (0.5 ml) of thc lcucocytc suspension and the Cnndida albicans solution were incubatcd for 30 mill at 37°C and 0.1 nil of the EDTA/salinc solution was then addcd to prevent clumping of tlie cells. Thc mixture was centrifuged gently for 5 inin and aftcr the supcriiatant had been removed a smear was made of the resulting cell deposit. The smears were stained with JennerGiemsa, examined microscopically and the number of Candida albicuris particles ingested by 200 neutropliils recorded. The phagocytic index (PI) is the average number of particles ingested per cell. The control for each time interval and cadi drug was taken as 100% PI and given the value I . The fall in PI for each drug was recorded as a percentage of the control value. The PI was estimated by one author and re-estimated blind by the other two authors. The differences between the three obscrvatioiis wcrc not statistically significant. EJect of time on the yhagocytic index. A freshly prepared pooled lcucocytc suspension was incubated at 37°C. At 1 5 miii intervals, for 90 min, I nil samples were removed after mixing and tlie phagocytic index of thc ncutropliils in each sample was measurcd. The results (Table 11) showed a fall in phagocytic index with incubation time. All subsequent estiniations were performed at a standard incubation time of 3 0 miii. Efect ofdrugs on the phugocytic index. Drug solutions were freshly prepared and 0. I nil of each solution incubated with 0.9 ml of the pooled leucocytc suspension at 37" C. Drug-incubation TABLE I. Thc effect of anti-inflammatory and cytotoxic agents on thc phagocytosis of ncutrophil leucocytes in vitro
Drug Phenylbutazone Acetylsalicylic acid Vincristine Vinblastine Cytosine arabinoside Busulphan Daunorubicin Mustine hydrochloride Procarbazine
Drng concentration (molar) x I0 -4 x I0 -3 5 x I0 2 x I0 - 5 I x I0 - - 5 I x I0 -I5 I x I0 --I5 I
2.5
I X I O - ~
I X I O - ~
Phagocytic index (% control value) atfonr incribation tiines t 3 0 wzin
15 rnin 96+
1.0
9SfO.S 78.5 0.9*
+
86 f0.6 89kO.S
65 f I.S* 66 f 0.6* 90+ 1.0 95.SkI.I
9IkO.S 84+1.5* 69.5 + I.O* 73 +0.2* 77+ I.2* 67+1.3* 73f2.I* 92 2.2 93 1.9
+
45 niin 81&1.25* 8 4 5 1.3* 62+1.1* 73 f O . S * 75.5 +0.5* 7IkI.5* 72.5+0.5* 95 f 1.2 Wfo.9
60 min
_~
71+1.3* 79 k 0.4* 54*0.5* 77tr.4* 75 +o.6* 74fI.I* 7 0 5 I.O* 99kO.5 9950.6
All observations are the results of three cxpcriments compared with three controls.
* Difference from controls significant (P
The Effect of Cytotoxic and Anti-inflammatory Drugs on the Phagocytosis, of Neutrophil Leucocytes J. A. WHITTAKER, H. R. HUGHES AND M. KHURSHID Departwent of Haenzatology, W e l s h Natioiial School oJ’M&iizc, C a r d i f (Received 22 April 1974; ncccptedfor pirblicntioii 3 J
i m
1974)
SUMMARY. The phagocytosis of hcat-killcd Candida albicarzs by iicutrophil lcucocytes was inhibited irz vitro by phciiylbutazonc and acctylsalicylic acid and by the cytotoxic drugs viiicristine, vinblastinc, cytosiiic arabinoside, dauiiorubiciii and busulphan, but not by mustiiic liydrochloridc or procarbazinc. In vivo studies in man havc shown a significant reduction in pliagocytosis after plicnylbutazoiic ingestion, but not following an oral dose of aspirin. Possible explanatory meclianisms of drug action on neutrophil phagocytosis arc discusscd. Sevcral chemically unrelated drugs have been sliowii to depress phagocytosis iii vitro. These iiiclude the anti-inflammatory drug phenylbutazonc (Straws ct a/, 1968; Ruutu & Kosunen, 1972) and tlic phciiothiazincs (Ruutu, 1972). A variety of cytotoxic agents is now in use for the treatment of maligiiaiit disease but many of these agents cause iieutropciiia and their usc is associatcd with an increased incidence of infection. In the prcseiit study we havc measured tlic cffcct of scveral cytotoxic agents 011 neutrophil phagocytic function and attempted to cor rclatc tlic known iir vifro depression of yhagocytosis caused by the anti-iiiflanimatory drugs phciiylbutazonc and acetylsalicylic acid with their effect iit vivo in human subjects. MATERIALS A N D METHODS
Drugs. The concentration of drugs uscd for itl vitro cxpcriiiiciits (Tablc I) was cquivalciit to coiiceiitratioiis achieved iii vivo by therapeutic dosage. Fresh solutions of each drug wcrc prepared immcdiatcly bcforc each cxpcriinciit and 0 .I 1111 of thc first drug solution dissolved in distillcd water or otlicr appropriatc solvc~itwas incubated with 0.9 1111 of thc lcucocytc suspciisioii. CoiitroI cultures wcrc prepared excluding tlic drugs but iiicludiiig thc solvent uscd to dissolve thc drug. Mdia arid reagents. (i) Hanks balaiiccd salt solution (HEJSS) without antibiotics. (ii) A heatkillcd suspension of Caridida albicam, 6 x 106 cclls/ml. (iii) EDTA solution 0.9% in sodium cliloridc 0.9%. (iv) Sterile 6”/, dcxtraii (in01 wt 150 000) in 0.9% sodium chloride. Preparation qf lezrcocytc strspcmion. 20 1111 vciious blood samples wcrc collcctcd with plastic syriiigcs from five hacmatologically noriii.al voluiitccrs into plastic bottlcs containing lithium heparin (Staync Laboratories Ltd) a i d 0.5 nil of dcxtraii/salinc solutioii. The red cells were Correspondence: Dr J. A. Whittnkcr, Dcpartnicii t o f Hncinatology, Wcluh Natioiial School of Mcdiciiic, I-lcath Park, Cardiff CF4 4XW.
273
J. A. Whittaker, H. R. Hughes and Ad. Khtdrshid
274
allowed to sediment at room temperature for 30 iiiiii and the leucocyte rich supernatants harvested, pooled and Centrifuged. The leucocytc deposit was washed twice in HBSS and finally resuspeiidcd in HBSS to give a final concciitratioii of leucocytes of 5 x 106 cells/ml. The superiiataiit plasma from the pooled fractioiis was saved and used to resuspend the leucocytes after incubation with thc drugs. Measrircnieiit of the ykugocytic indcx. The pliagocytosis of Curidida albicuns particles was measured by a modification of thc method of Millcr & Nilsson (1970). Equal volumes (0.5 ml) of thc lcucocytc suspension and the Cnndida albicans solution were incubatcd for 30 mill at 37°C and 0.1 nil of the EDTA/salinc solution was then addcd to prevent clumping of tlie cells. Thc mixture was centrifuged gently for 5 inin and aftcr the supcriiatant had been removed a smear was made of the resulting cell deposit. The smears were stained with JennerGiemsa, examined microscopically and the number of Candida albicuris particles ingested by 200 neutropliils recorded. The phagocytic index (PI) is the average number of particles ingested per cell. The control for each time interval and cadi drug was taken as 100% PI and given the value I . The fall in PI for each drug was recorded as a percentage of the control value. The PI was estimated by one author and re-estimated blind by the other two authors. The differences between the three obscrvatioiis wcrc not statistically significant. EJect of time on the yhagocytic index. A freshly prepared pooled lcucocytc suspension was incubated at 37°C. At 1 5 miii intervals, for 90 min, I nil samples were removed after mixing and tlie phagocytic index of thc ncutropliils in each sample was measurcd. The results (Table 11) showed a fall in phagocytic index with incubation time. All subsequent estiniations were performed at a standard incubation time of 3 0 miii. Efect ofdrugs on the phugocytic index. Drug solutions were freshly prepared and 0. I nil of each solution incubated with 0.9 ml of the pooled leucocytc suspension at 37" C. Drug-incubation TABLE I. Thc effect of anti-inflammatory and cytotoxic agents on thc phagocytosis of ncutrophil leucocytes in vitro
Drug Phenylbutazone Acetylsalicylic acid Vincristine Vinblastine Cytosine arabinoside Busulphan Daunorubicin Mustine hydrochloride Procarbazine
Drng concentration (molar) x I0 -4 x I0 -3 5 x I0 2 x I0 - 5 I x I0 - - 5 I x I0 -I5 I x I0 --I5 I
2.5
I X I O - ~
I X I O - ~
Phagocytic index (% control value) atfonr incribation tiines t 3 0 wzin
15 rnin 96+
1.0
9SfO.S 78.5 0.9*
+
86 f0.6 89kO.S
65 f I.S* 66 f 0.6* 90+ 1.0 95.SkI.I
9IkO.S 84+1.5* 69.5 + I.O* 73 +0.2* 77+ I.2* 67+1.3* 73f2.I* 92 2.2 93 1.9
+
45 niin 81&1.25* 8 4 5 1.3* 62+1.1* 73 f O . S * 75.5 +0.5* 7IkI.5* 72.5+0.5* 95 f 1.2 Wfo.9
60 min
_~
71+1.3* 79 k 0.4* 54*0.5* 77tr.4* 75 +o.6* 74fI.I* 7 0 5 I.O* 99kO.5 9950.6
All observations are the results of three cxpcriments compared with three controls.
* Difference from controls significant (P
