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Failure of water to lyse polymorphonuclear neutrophils completely
289
Journal of Immunological Methods, 124 (1989) 289-291 Elsevier JIM 05410
Letter to the editors
Failure of water to lyse polymorphonuclear neutrophils completely Role of pH and implications for assessment of bacterial killing R.A. Gargan, W. Brumfitt and J.M.T. Hamilton-Miller Department of Medical Microbiology, The Royal Free Hospital and School of Medicine, Pond Street, Hampsteaa~London NW3 2QG, U.K. (Received 21 July 1989, revised received 7 September 1989, accepted 11 September 1989)
Dear Editors, For bacterial killing experiments most workers lyse polymorphonuclear neutrophils (PMNs) by osmotic shock in distilled water, either at room temperature (Babior and Cohen, 1981) or at 0 °C (Leijh et al., 1986). This method of lysis appears to be the least harmful to intracellular microorganisms. However, complete lysis of PMNs can be difficult to achieve: Hansen and Andersen (1973) showed that rapid freezing and thawing up to six times achieved little more than 50% lysis. In view of the report by Triger and Smith (1966) that low osmolality, together with raised pH and temperature caused the lysis of PMNs in urine. We have investigated the optimum pH and temperature of distilled water required to totally lyse PMNs and the effects of partial lysis of PMNs on the subsequent calculation of intracellular bacterial killing. Ten samples of 4 ml EDTA anticoagulated venous blood were taken from four healthy individuals. PMNs were routinely isolated by differential centrifugation after lysis of red cells with isotonic ammonium chloride (Eggleton et al., 1989), and resuspended in Hanks' balanced salt solution (HBSS) (Gibco, Scotland). The dextran sedimentation method for separation of the PMNs was as outlined by Babior and Cohen (1981).
Correspondence to: R.A. Gargan, Department of Medical Microbiology, The Royal Free Hospital and School of Medicine, Pond Street, Hampstead, London NW3 2QG, U.K.
Sterile pyrogen-free distilled water (Travenol, U.K.) pH 5.3 as purchased, was adjusted to pH values 7, 9, 10 and 11 with NaOH. 20 #1 volumes of the PMN suspensions were added to 1 ml distilled water to give approximately 5 × 105 PMNs/ml at the different pHs and held at 37 o C, 21°C and 0 ° C for 5 min. HBSS at 0 ° C acted as the 100% control (20 #1 of HBSS did not alter the pH of the 1 ml of water). Counts at pH 5.3, 7 and 9 were made in duplicate with a Coulter counter (model ZB1) and at pH values 10 and 11 with a counting chamber (due to very low counts). Cell viability was determined using the ethidium bromide technique of Ford (1978). For phagocytosis, an overnight growth of Staphylococcus aureus (NCTC 6571) in brain-heart infusion broth (Oxoid, U.K.) was washed, diluted in HBSS and opsonised with 10% serum at 37 °C for 30 rain. This was added to the PMNs to give a bacteria to PMN ratio of 10 : 1 and incubated for 15 min at 37 ° C. 20 #1 of this reaction mixture was added to 1 ml water for 5 rain at the pH values and temperatures described and the PMNs counted. For bacterial killing the same strain of S. aureus, two strains of S. epidermidis and an Escherichia coli were grown and opsonised as above. 200 /~1 of bacteria were mixed with the same volume of PMNs to give the ratio as described. This was immediately sampled by removing 50 #1 to 4.95 ml water at pH 11 for 5 rain at 37°C for duplicate viable counts. After rotating for 1 h at 37 °C the mixture was sampled as before with an additional 50 #1 removed to 4.95 ml water at pH 5.3 for 5 min at 0°C. Bacterial killing was ex-
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
290 100
PF7
80 PEREENT PMN
60
SURVIVAL 40
20
\. 7
9
11
7
9
WATER
11
7
9
11
pH
Fig. 1. Effect of water pH on PMN lysis at: (a) 0 o C, (b) 21°C and (c) 37 o C. The solid line joins the mean values and the vertical bars indicate standard errors (n = 10).
pressed as a percentage reduction relative to the initial viable count. All the organisms were unaffected by 10% serum. Stained slides of the preparations confirmed > 95% phagocytosis. PMNs were very resistant to lysis by osmotic shock for 5 rain at pH 5.3. Raising the pH of the water from 5.3 to 9 had little effect on their lysis, but increasing the temperature at pH 5.3 reduced their survival from 86% at 0 ° C to 69% at 3 7 ° C (Figs. l a - l c ) . However, changing the p H to 10 or 11 had a profound effect, with an increase of temperature enhancing lysis. At pH 10, 44, 29 and 13% of PMNs survived at 0 ° C , 21°C and 3 7 ° C respectively, while at pH 11 only 4, 2 and 0.5% PMNs survived. N o lysis of PMNs took place in HBSS controls adjusted from pH 7 to 11. All PMNs surviving osmotic lysis were found to fluoresce red indicating damaged membranes. In contrast control PMNs from HBSS fluoresced green. Cells surviving osmotic shock at p H values 5 and 7 were 74% (SD + 3.8, n = 9) PMNs, so the smaller non-PMN population present in the original cell preparations had not disproportionately resisted lysis. The nuclei of surviving cells at pH 9 and above became distorted and did not allow differentiation. As phagocytosis is known to cause changes to the PMN membrane, we investigated the effect of
osmotic shock on PMNs that had previously phagocytosed opsonised S. aureus. However, PMN disruption closely followed the pattern of Figs. l a - l c , with complete lysis only at pH 11 at 37 o C. PMNs isolated by dextran sedimentation also behaved in a similar way. The use of non-sterile double distilled water (pH 5.6) or deionised water (pH 5.3) similarly did not influence the results. Incomplete lysis of PMNs that had phagocytosed bacteria (confirmed by microscopy) should result in bacteria not being released from intact PMNs. This was verified by viable counts which
TABLE I FALSELY RAISED BACTERIALKILLING CAUSED BY PARTIAL PMN LYSIS. Organism
% kill when PMNs lysed at: Differenceb in % kill pH 11, pH 5.3, 37oC a 0oC a
S. aureus S. epidermidis
70.5
80.7
10.2
(strain 1023) 77.4
82.5
5.1
50.9 72.2
11.3 17
S. epidermidis
(strain 5970) 39.6 55.2
E. coli
a Mean value (n = 3). b Difference in apparent killing at pH 11 and pH 5.3.
291 were significantly lower after P M N s were h e l d at p H 5.3, in c o m p a r i s o n to those held at p H 11 ( T a b l e I, P < 0.005, W i l c o x o n ' s m a t c h e d p a i r s s i g n e d - r a n k test). This led to a n e r r o n e o u s l y high e s t i m a t e of killing b y P M N s , which, overall, averaged 11% (range 0-25%). T h e s t a p h y l o c o c c i were n o t killed b y p H values > 11, in the u n o p s o n i s e d a n d o p s o n i s e d state. However, care h a d to b e exercised to ensure t h a t the p H of the w a t e r d i d n o t exceed 11 for the o p s o n i s e d E. coli, which p r o v e d to b e sensitive to a p H of 11.2 a n d above. It is c o n c l u d e d that a highly effective w a y to lyse P M N s w i t h o u t d a m a g i n g intraceUular b a c t e r i a is to use distilled water at a raised p H at 37 ° C.
References Babior, B.M. and Cohen, H.J. (1981) Measurement of neutrophil function; phagocytosis, degranulation, the respira-
tory burst and bacterial killing: In: M.J. Cline (Ed.), Methods in Haematology, Vol. 3. Churchill-Livingstone, New York, p. 1. Eggleton, P., Gargan, R. and Fisher, D. (1989) Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers. J. Immunol. Methods 121, 105. Ford, W.L. (1978) The preparation and labelling of lymphocytes. In: D.M. Weir (Ed.), Handbook of Experimental Immunology, Vol. 2. Blackwell Scientific Publication, Oxford, p. 23.10. Hansen, N.E. and Andersen, V. (1973) Lysozyme activity in human neutrophilic granulocytes. Br. J. Haematol. 24, 613. Leijh, P.C.J., Van Furth, R. and Van Zwet, T.L. (1986) In vitro determination of phagocytosis and intracellular killing by polymorphonuclear and mononuclear phagocytes. In: D.M. Weir (Ed.), Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford, p. 46.1. Triger, D.R. and Smith, J.W.G. (1966) Survival of urinary leucocytes. J. Clin. Pathol. 19, 443.
Journal of Immunological Methods, 124 (1989) 289-291 Elsevier JIM 05410
Letter to the editors
Failure of water to lyse polymorphonuclear neutrophils completely Role of pH and implications for assessment of bacterial killing R.A. Gargan, W. Brumfitt and J.M.T. Hamilton-Miller Department of Medical Microbiology, The Royal Free Hospital and School of Medicine, Pond Street, Hampsteaa~London NW3 2QG, U.K. (Received 21 July 1989, revised received 7 September 1989, accepted 11 September 1989)
Dear Editors, For bacterial killing experiments most workers lyse polymorphonuclear neutrophils (PMNs) by osmotic shock in distilled water, either at room temperature (Babior and Cohen, 1981) or at 0 °C (Leijh et al., 1986). This method of lysis appears to be the least harmful to intracellular microorganisms. However, complete lysis of PMNs can be difficult to achieve: Hansen and Andersen (1973) showed that rapid freezing and thawing up to six times achieved little more than 50% lysis. In view of the report by Triger and Smith (1966) that low osmolality, together with raised pH and temperature caused the lysis of PMNs in urine. We have investigated the optimum pH and temperature of distilled water required to totally lyse PMNs and the effects of partial lysis of PMNs on the subsequent calculation of intracellular bacterial killing. Ten samples of 4 ml EDTA anticoagulated venous blood were taken from four healthy individuals. PMNs were routinely isolated by differential centrifugation after lysis of red cells with isotonic ammonium chloride (Eggleton et al., 1989), and resuspended in Hanks' balanced salt solution (HBSS) (Gibco, Scotland). The dextran sedimentation method for separation of the PMNs was as outlined by Babior and Cohen (1981).
Correspondence to: R.A. Gargan, Department of Medical Microbiology, The Royal Free Hospital and School of Medicine, Pond Street, Hampstead, London NW3 2QG, U.K.
Sterile pyrogen-free distilled water (Travenol, U.K.) pH 5.3 as purchased, was adjusted to pH values 7, 9, 10 and 11 with NaOH. 20 #1 volumes of the PMN suspensions were added to 1 ml distilled water to give approximately 5 × 105 PMNs/ml at the different pHs and held at 37 o C, 21°C and 0 ° C for 5 min. HBSS at 0 ° C acted as the 100% control (20 #1 of HBSS did not alter the pH of the 1 ml of water). Counts at pH 5.3, 7 and 9 were made in duplicate with a Coulter counter (model ZB1) and at pH values 10 and 11 with a counting chamber (due to very low counts). Cell viability was determined using the ethidium bromide technique of Ford (1978). For phagocytosis, an overnight growth of Staphylococcus aureus (NCTC 6571) in brain-heart infusion broth (Oxoid, U.K.) was washed, diluted in HBSS and opsonised with 10% serum at 37 °C for 30 rain. This was added to the PMNs to give a bacteria to PMN ratio of 10 : 1 and incubated for 15 min at 37 ° C. 20 #1 of this reaction mixture was added to 1 ml water for 5 rain at the pH values and temperatures described and the PMNs counted. For bacterial killing the same strain of S. aureus, two strains of S. epidermidis and an Escherichia coli were grown and opsonised as above. 200 /~1 of bacteria were mixed with the same volume of PMNs to give the ratio as described. This was immediately sampled by removing 50 #1 to 4.95 ml water at pH 11 for 5 rain at 37°C for duplicate viable counts. After rotating for 1 h at 37 °C the mixture was sampled as before with an additional 50 #1 removed to 4.95 ml water at pH 5.3 for 5 min at 0°C. Bacterial killing was ex-
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
290 100
PF7
80 PEREENT PMN
60
SURVIVAL 40
20
\. 7
9
11
7
9
WATER
11
7
9
11
pH
Fig. 1. Effect of water pH on PMN lysis at: (a) 0 o C, (b) 21°C and (c) 37 o C. The solid line joins the mean values and the vertical bars indicate standard errors (n = 10).
pressed as a percentage reduction relative to the initial viable count. All the organisms were unaffected by 10% serum. Stained slides of the preparations confirmed > 95% phagocytosis. PMNs were very resistant to lysis by osmotic shock for 5 rain at pH 5.3. Raising the pH of the water from 5.3 to 9 had little effect on their lysis, but increasing the temperature at pH 5.3 reduced their survival from 86% at 0 ° C to 69% at 3 7 ° C (Figs. l a - l c ) . However, changing the p H to 10 or 11 had a profound effect, with an increase of temperature enhancing lysis. At pH 10, 44, 29 and 13% of PMNs survived at 0 ° C , 21°C and 3 7 ° C respectively, while at pH 11 only 4, 2 and 0.5% PMNs survived. N o lysis of PMNs took place in HBSS controls adjusted from pH 7 to 11. All PMNs surviving osmotic lysis were found to fluoresce red indicating damaged membranes. In contrast control PMNs from HBSS fluoresced green. Cells surviving osmotic shock at p H values 5 and 7 were 74% (SD + 3.8, n = 9) PMNs, so the smaller non-PMN population present in the original cell preparations had not disproportionately resisted lysis. The nuclei of surviving cells at pH 9 and above became distorted and did not allow differentiation. As phagocytosis is known to cause changes to the PMN membrane, we investigated the effect of
osmotic shock on PMNs that had previously phagocytosed opsonised S. aureus. However, PMN disruption closely followed the pattern of Figs. l a - l c , with complete lysis only at pH 11 at 37 o C. PMNs isolated by dextran sedimentation also behaved in a similar way. The use of non-sterile double distilled water (pH 5.6) or deionised water (pH 5.3) similarly did not influence the results. Incomplete lysis of PMNs that had phagocytosed bacteria (confirmed by microscopy) should result in bacteria not being released from intact PMNs. This was verified by viable counts which
TABLE I FALSELY RAISED BACTERIALKILLING CAUSED BY PARTIAL PMN LYSIS. Organism
% kill when PMNs lysed at: Differenceb in % kill pH 11, pH 5.3, 37oC a 0oC a
S. aureus S. epidermidis
70.5
80.7
10.2
(strain 1023) 77.4
82.5
5.1
50.9 72.2
11.3 17
S. epidermidis
(strain 5970) 39.6 55.2
E. coli
a Mean value (n = 3). b Difference in apparent killing at pH 11 and pH 5.3.
291 were significantly lower after P M N s were h e l d at p H 5.3, in c o m p a r i s o n to those held at p H 11 ( T a b l e I, P < 0.005, W i l c o x o n ' s m a t c h e d p a i r s s i g n e d - r a n k test). This led to a n e r r o n e o u s l y high e s t i m a t e of killing b y P M N s , which, overall, averaged 11% (range 0-25%). T h e s t a p h y l o c o c c i were n o t killed b y p H values > 11, in the u n o p s o n i s e d a n d o p s o n i s e d state. However, care h a d to b e exercised to ensure t h a t the p H of the w a t e r d i d n o t exceed 11 for the o p s o n i s e d E. coli, which p r o v e d to b e sensitive to a p H of 11.2 a n d above. It is c o n c l u d e d that a highly effective w a y to lyse P M N s w i t h o u t d a m a g i n g intraceUular b a c t e r i a is to use distilled water at a raised p H at 37 ° C.
References Babior, B.M. and Cohen, H.J. (1981) Measurement of neutrophil function; phagocytosis, degranulation, the respira-
tory burst and bacterial killing: In: M.J. Cline (Ed.), Methods in Haematology, Vol. 3. Churchill-Livingstone, New York, p. 1. Eggleton, P., Gargan, R. and Fisher, D. (1989) Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers. J. Immunol. Methods 121, 105. Ford, W.L. (1978) The preparation and labelling of lymphocytes. In: D.M. Weir (Ed.), Handbook of Experimental Immunology, Vol. 2. Blackwell Scientific Publication, Oxford, p. 23.10. Hansen, N.E. and Andersen, V. (1973) Lysozyme activity in human neutrophilic granulocytes. Br. J. Haematol. 24, 613. Leijh, P.C.J., Van Furth, R. and Van Zwet, T.L. (1986) In vitro determination of phagocytosis and intracellular killing by polymorphonuclear and mononuclear phagocytes. In: D.M. Weir (Ed.), Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford, p. 46.1. Triger, D.R. and Smith, J.W.G. (1966) Survival of urinary leucocytes. J. Clin. Pathol. 19, 443.