M I G R A T I O N O F PERITONEAL POLYMORPHONUCLEAR LEUCOCYTES I N T H E R A T
FIONA M. CUNNINGHAM, M. J. H. SMITH,A. W. FORD-HUTCHINSON AND J. R. WALKER Biochemical Pharmacology Research Unit, Department of Chemical Pathology, King’s College Hospital Medical School, Denmark Hill,London, SE.5 8RX
POLYM O R P H O N U C L E A R leucocytes (PMNs), prepared from peritoneal exudates rather than from the circulation, have been widely used for the study of many aspects of leucocyte function and behaviour. This is because the peritoneal cells are more readily obtainable in large numbers as a relatively pure population. Responses of both circulating and elicited PMNs are qualitatively similar although they may not be identical. Thus it has been reported that they differ with respect to the directed migration induced by prostaglandin El in vitro (Walker, Smith and Ford-Hutchinson, 1976). We have therefore studied : first, the in-vivo movement of radio labelled peritoneal exudate cells into implanted sponges; secondly, the effects of intravenous administration of peritoneal exudate cells on the accumulation of leucocytes into implanted sponges; and, finally, the chemotaxis of peritoneal and peripheral PMNs in vitro. MATERIALS AND METHODS Animals Female albino rats (1 50/200g) were obtained from Oxfordshire Laboratory Animal Colonies, 1976, Ltd., Porton Wistar/OLA outbred rats being used throughout except for the experiments using inbred rats in which either WAG/OLA or F344/OLA inbred rats were used. Cell suspensions Peritoneal exudates were induced in rats by the intra-peritoneal injection of either 6 ml of 12 per cent. sodium caseinate, 20 ml of 2 per cent, starch or 20 ml of 0.1 per cent. oyster glycogen. After 24 hr the animals were injected intra-peritoneally with 20 ml of Hanks Balanced Salt Solution (HBSS) containing 5 units/ml heparin. The animals were killed after 45 sec and a small incision was made in the abdomen from which the peritoneal exudate was removed. The cells were centrifuged (100 g, 10 min.), washed once in HBSS and were then resuspended at a concentration of 4 x lO7/ml in Medium 199 containing 25 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulphonicacid) buffer, pH 7.3, and 20 per cent. heat inactivated foetal calf serum. The following standard methods were used to assess cell viability and phagocytic activity; trypan blue dye exclusion, the release of the cytoplasmic enzyme lactate dehydrogenase (LHD) and the ability of the cells to phagocytose zymosan particles as measured by the release of ,%glucuronidase. The 8glucuronidase results have been expressed as Sigma units. One Sigma unit will liberate 10 pg of phenolphthalein from phenolphthalein glucuronide per hour at 37°C. Acceptedfor publication 5 Sep. 1978. 1. PATH.-VOL.
128 (1979)
15
16
CUNNINGHAM, SMITH, FORD-HUTCHINSON AND WALKER
Radioactive labelling Cell suspensions were labelled with sodium chromate (51Cr) in sterile isotonic saline (Radiochemical Centre, Amersham) as described by Perper et al. (1974). After washing, peritoneal exudate cells were resuspended in Medium 199 at concentrations of 5.5, 5.9 and 3.5 x lO’/ml respectively for starch, sodium caseinate and glycogen elicited preparations. Radioactive analyses were carried out with a LKB Wallac 8000 gamma counter. Samples were counted for 5 min., and the results expressed as c.p.m. with the background counts subtracted. Sponge implantation The model used to study the accumulation of labelled and non-labelled elicited peritoneal cells in vivo was the sponge implantation technique of Ford-Hutchinson, Walker and Smith (1978) using six sponges per rat. Accumulation of 5 1Cr labelled peritoneal cells into implanted sponges Groups, each of five rats, were injected intravenously with 1 ml of 5lCr labelled peritoneal exudate cells at the same time as sponge implantation. After 1 , 5 or 9 hr, 1 ml blood samples were obtained by cardiac puncture, the animals were killed and the sponges removed. Three sponges from each rat were combined and the total radioactivity per sponge determined. The exudate was removed from the remaining three sponges, pooled and separated into supernatant, cell (100 g, 2 x 10 min.) and cell debris fractions (100,000 g, 1 hr). Radioactivity in each fraction was determined. A 0.1 ml aliquot of whole blood was centrifuged and the radioactivity in the cell and plasma fractions was determined. From these results it was possible to estimate the number of peritoneal PMNS entering the sponge exudate. Accumulation of free 5 1Cr into implanted sponges Groups, each of five rats, were injected intravenously with 1 ml of 51Cr solution (2pCi/ml) in Medium 199 mixed with lmg of ascorbic acid per 10 pCi. Asorbic acid reduces chromate ions from the hexavalent to the trivalent state and, as the 51Cr ion either does not pass through the plasma membrane or has a limited passage, (Mertz, 1969) the in-vivo labelling of cells is prevented. Sponges were implanted in the rats for 1, 5 or 9 hr and the radioactivity in the sponge exudate and the blood measured as described above. Effects of intravenousadministrationofperitoneal cells on leucocyte accumulation into implanted sponges Groups, each of five outbred or inbred rats, were injected intravenously with either 1 mI of peritoneal exudate cells (7 x lo7 celIs/mI) elicited by sodium caseinate or 1 ml of Medium 199, and sponges were implanted for 5 hr. Leucocyte accumulation within the sponge exudates was assessed by light microscopy following trypsinisation of the sponges (Ford-Hutchinson et al.). Chemotaxis in vitro Chemotaxis assays in vitro were performed using Adaps disposable chemotactic chambers (Adaps, Inc., Dedham, Mass., USA) and 5 pm millipore filters. The chambers were incubated for 2 hr at 37°C in a humid atmosphere, the filters were removed and stained with Erlich’s haematoxylin. The results are expressed as the number of cells (per high power field x 500) which had migrated completely through the filter. Peripheral, peritoneal and labelled peritoneal cells were suspended as a concentration of 2.5 x 106 cells/ml in Eagle’s Minimum Essential Medium buffered to pH 7.3 with 25 mM HEPES. Peripheral rat cells were obtained by dextran sedimentation according to the directions of Cunningham et al. (1978). Chemotactic factors were generated by incubation of fresh serum with 500 pg/ml washed, boiled zymosan at 37°C for 1 hr followed by heat inactivation (56”C, 45 min.). The zymosan was removed from the activated serum by centrifugation prior to use.
17
POL YMORPH MIGRA TI0N
RESULTS Cell viability and phagocytic capability of peritoneal exudate cells All three exudates contained greater than 80 per cent. PMNs which were more than 98 per cent. viable as assessed by trypan blue exclusion. Increased release of /3-glucuronidase on exposure to zymosan showed that the PMNs from all three exudates were capable of active phagocytosis. There was no corresponding loss of cell viability during phagocytosis as shown by LDH levels and trypan blue exclusion. Sodium caseinate elicited peritoneal cells were used throughout the work, except where otherwise stated. This is because the total cell numbers in this exudate were more than twice those obtained using either starch or glycogen. Mean total cell numbers were 1 . 7 108, ~ 6 . 7 107 ~ and 5 . 2 ~ 107 cells per rat for sodium caseinate, starch and glycogen elicited exudates respectively. Radioactivity labelling of exudate cells In the experiments in which peritoneal cells were labelled with 5 0 , greater than 95 per cent. of the 5lCr was associated with the washed cells which were more than 94 per cent. viable as shown by trypan blue exclusion, and retained full phagocytic activity. Accumulation of 5 0 in sponge exudates Over a 9 hr period there was an increase in c.p.m. associated with the cell fraction of sponge exudates from rats injected intravenously with 51Cr labelled peritoneal cells (table I). This radioactivity, however, accounts for less than 10 per cent. of the total 51Cr with more than 89 per cent. being TABLEI The distribution of radioactivity in I , 5 and 9 hr sponges from rats injected intravenously with sodium caseinate elicited peritoneal PMNs Time hrs. 1 5 9
No. of
animals 10 10 10
Total
c.p.m.1
sponge
103.212275 162.1 h19.7 237.01t25.5
Percentage of total c.p.m. A
Cell debris 0.1 0.9 1.2
Cells 1.1 4.9
9.4
Supernatant 98.8
,
94.2 89.4
found as free 51Cr in the supernatant fraction. It was calculated that 1.2 x 105 exudate cells/ml migrated into 5 hr sponges. A similar distribution of 51Cr was found in 5 hr sponge exudates following the intravenous injection of labelled exudate cells elicited by either starch or glycogen. The intravenous injection of reduced 51Cr in Medium 199, in an amount equivalent to the steady state level of plasma radioactivity in rats injected intravenously with labelled peritoneal exudate cells, was used to determine the proportion of the free 51Cr in the supernatant fraction of sponge exudates which could be accounted for by 51Cr free in the plasma (see Perper e f al.). Table I1 shows the c.p.m. in the supernatant of sponge exudates expressed as equivalent
18
CUNNINGHAM, SMITH, FORD-HUTCHINSON AND WALKER
TABLE I1 Radiochromium in supernatants of sponge exudates expressed as equivalent mls of plasma Time of sponge implantation
(W 1 5 9
,
Source of 5lCr
Reduced 5lCr solution (i.v.) 0.07 0.15 022
L
Labelled peritoneal cells (i.v.) 0.06 0.13 0.23
mls of plasma. These results suggest that after intravenous injection of 51Cr labelled cells or reduced 51Cr all the radioactivity in the supernatant fraction could be accounted for by plasma leakage. No significant counts were found in the circulating cells after the injection of reduced 51Cr. EfSect of peritoneal cells on accumulation of leucocytes into 5 hr sponge exudates Intravenous adminstration of peritoneal exudate cells produce a significant reduction in the number of cells accumulating in 5 hr sponge exudates. This effect was seen in both the outbred Wistar rats used throughout the work and in the two strains of inbred rats (table 111). TABLEI11 Leucocyte accumulation in 5 hr sponge exudates Rat strain n Control Test 6.9f0.1* Outbred Wistar/OLAC 10 21.0*2.5 5 8.4f0.06 4.0f0.05* Inbred WAGlOLAC 0.8f0.06* Inbred F344/OLAC 5 3.6f002 Results expressed as means *S.E. x 105/m1. Control rats injected with 1 ml Medium 199; test rats injected with sodium caseinate-induced peritoneal exudate cells. * P < 0.005 compared with corresponding control.
Chemotaxis in vitro Peripheral, peritoneal and labelled peritoneal cells showed a significant chemotactic response to serum generated chemotactic factors in vitro. The peritoneal cell response was significantly less than that of the peripheral cells (table IV).
DISCUSSION Peritoneal exudate cells labelled with 51Cr have been used for the study of chemotaxis in vitro (Gallin, Clark and Kimball, 1973) and the accumulation of leucocytes at inflammatory sites in vivo (Perper et al.; Jones, Richardson and Kay, 1977). In experiments utilising 51Cr labelled peritoneal cells for in-vivo studies it has been assumed, first, that the radioactivity accumulating at the inflammatory site is accounted for by the presence of labelled cells that have emigrated out of the vasculature into the inflammatory exudate and secondly, that the peritoneal cells behave in an identical fashion to the circulating cell population.
POLYMORPH MIGRA TION
19
TABLE IV Comparison of the in-vitro chemotaxis of peripheral and sodium caseinate induced peritoneal neutrophils Cell source n Directed migration? 15.3f2.3 Peripheral 8 Peritoneal 8 7.7*0.88* 7.2+0.67* Labelled peritoneal 8 Results expressed as means +S.E. * P
FIONA M. CUNNINGHAM, M. J. H. SMITH,A. W. FORD-HUTCHINSON AND J. R. WALKER Biochemical Pharmacology Research Unit, Department of Chemical Pathology, King’s College Hospital Medical School, Denmark Hill,London, SE.5 8RX
POLYM O R P H O N U C L E A R leucocytes (PMNs), prepared from peritoneal exudates rather than from the circulation, have been widely used for the study of many aspects of leucocyte function and behaviour. This is because the peritoneal cells are more readily obtainable in large numbers as a relatively pure population. Responses of both circulating and elicited PMNs are qualitatively similar although they may not be identical. Thus it has been reported that they differ with respect to the directed migration induced by prostaglandin El in vitro (Walker, Smith and Ford-Hutchinson, 1976). We have therefore studied : first, the in-vivo movement of radio labelled peritoneal exudate cells into implanted sponges; secondly, the effects of intravenous administration of peritoneal exudate cells on the accumulation of leucocytes into implanted sponges; and, finally, the chemotaxis of peritoneal and peripheral PMNs in vitro. MATERIALS AND METHODS Animals Female albino rats (1 50/200g) were obtained from Oxfordshire Laboratory Animal Colonies, 1976, Ltd., Porton Wistar/OLA outbred rats being used throughout except for the experiments using inbred rats in which either WAG/OLA or F344/OLA inbred rats were used. Cell suspensions Peritoneal exudates were induced in rats by the intra-peritoneal injection of either 6 ml of 12 per cent. sodium caseinate, 20 ml of 2 per cent, starch or 20 ml of 0.1 per cent. oyster glycogen. After 24 hr the animals were injected intra-peritoneally with 20 ml of Hanks Balanced Salt Solution (HBSS) containing 5 units/ml heparin. The animals were killed after 45 sec and a small incision was made in the abdomen from which the peritoneal exudate was removed. The cells were centrifuged (100 g, 10 min.), washed once in HBSS and were then resuspended at a concentration of 4 x lO7/ml in Medium 199 containing 25 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulphonicacid) buffer, pH 7.3, and 20 per cent. heat inactivated foetal calf serum. The following standard methods were used to assess cell viability and phagocytic activity; trypan blue dye exclusion, the release of the cytoplasmic enzyme lactate dehydrogenase (LHD) and the ability of the cells to phagocytose zymosan particles as measured by the release of ,%glucuronidase. The 8glucuronidase results have been expressed as Sigma units. One Sigma unit will liberate 10 pg of phenolphthalein from phenolphthalein glucuronide per hour at 37°C. Acceptedfor publication 5 Sep. 1978. 1. PATH.-VOL.
128 (1979)
15
16
CUNNINGHAM, SMITH, FORD-HUTCHINSON AND WALKER
Radioactive labelling Cell suspensions were labelled with sodium chromate (51Cr) in sterile isotonic saline (Radiochemical Centre, Amersham) as described by Perper et al. (1974). After washing, peritoneal exudate cells were resuspended in Medium 199 at concentrations of 5.5, 5.9 and 3.5 x lO’/ml respectively for starch, sodium caseinate and glycogen elicited preparations. Radioactive analyses were carried out with a LKB Wallac 8000 gamma counter. Samples were counted for 5 min., and the results expressed as c.p.m. with the background counts subtracted. Sponge implantation The model used to study the accumulation of labelled and non-labelled elicited peritoneal cells in vivo was the sponge implantation technique of Ford-Hutchinson, Walker and Smith (1978) using six sponges per rat. Accumulation of 5 1Cr labelled peritoneal cells into implanted sponges Groups, each of five rats, were injected intravenously with 1 ml of 5lCr labelled peritoneal exudate cells at the same time as sponge implantation. After 1 , 5 or 9 hr, 1 ml blood samples were obtained by cardiac puncture, the animals were killed and the sponges removed. Three sponges from each rat were combined and the total radioactivity per sponge determined. The exudate was removed from the remaining three sponges, pooled and separated into supernatant, cell (100 g, 2 x 10 min.) and cell debris fractions (100,000 g, 1 hr). Radioactivity in each fraction was determined. A 0.1 ml aliquot of whole blood was centrifuged and the radioactivity in the cell and plasma fractions was determined. From these results it was possible to estimate the number of peritoneal PMNS entering the sponge exudate. Accumulation of free 5 1Cr into implanted sponges Groups, each of five rats, were injected intravenously with 1 ml of 51Cr solution (2pCi/ml) in Medium 199 mixed with lmg of ascorbic acid per 10 pCi. Asorbic acid reduces chromate ions from the hexavalent to the trivalent state and, as the 51Cr ion either does not pass through the plasma membrane or has a limited passage, (Mertz, 1969) the in-vivo labelling of cells is prevented. Sponges were implanted in the rats for 1, 5 or 9 hr and the radioactivity in the sponge exudate and the blood measured as described above. Effects of intravenousadministrationofperitoneal cells on leucocyte accumulation into implanted sponges Groups, each of five outbred or inbred rats, were injected intravenously with either 1 mI of peritoneal exudate cells (7 x lo7 celIs/mI) elicited by sodium caseinate or 1 ml of Medium 199, and sponges were implanted for 5 hr. Leucocyte accumulation within the sponge exudates was assessed by light microscopy following trypsinisation of the sponges (Ford-Hutchinson et al.). Chemotaxis in vitro Chemotaxis assays in vitro were performed using Adaps disposable chemotactic chambers (Adaps, Inc., Dedham, Mass., USA) and 5 pm millipore filters. The chambers were incubated for 2 hr at 37°C in a humid atmosphere, the filters were removed and stained with Erlich’s haematoxylin. The results are expressed as the number of cells (per high power field x 500) which had migrated completely through the filter. Peripheral, peritoneal and labelled peritoneal cells were suspended as a concentration of 2.5 x 106 cells/ml in Eagle’s Minimum Essential Medium buffered to pH 7.3 with 25 mM HEPES. Peripheral rat cells were obtained by dextran sedimentation according to the directions of Cunningham et al. (1978). Chemotactic factors were generated by incubation of fresh serum with 500 pg/ml washed, boiled zymosan at 37°C for 1 hr followed by heat inactivation (56”C, 45 min.). The zymosan was removed from the activated serum by centrifugation prior to use.
17
POL YMORPH MIGRA TI0N
RESULTS Cell viability and phagocytic capability of peritoneal exudate cells All three exudates contained greater than 80 per cent. PMNs which were more than 98 per cent. viable as assessed by trypan blue exclusion. Increased release of /3-glucuronidase on exposure to zymosan showed that the PMNs from all three exudates were capable of active phagocytosis. There was no corresponding loss of cell viability during phagocytosis as shown by LDH levels and trypan blue exclusion. Sodium caseinate elicited peritoneal cells were used throughout the work, except where otherwise stated. This is because the total cell numbers in this exudate were more than twice those obtained using either starch or glycogen. Mean total cell numbers were 1 . 7 108, ~ 6 . 7 107 ~ and 5 . 2 ~ 107 cells per rat for sodium caseinate, starch and glycogen elicited exudates respectively. Radioactivity labelling of exudate cells In the experiments in which peritoneal cells were labelled with 5 0 , greater than 95 per cent. of the 5lCr was associated with the washed cells which were more than 94 per cent. viable as shown by trypan blue exclusion, and retained full phagocytic activity. Accumulation of 5 0 in sponge exudates Over a 9 hr period there was an increase in c.p.m. associated with the cell fraction of sponge exudates from rats injected intravenously with 51Cr labelled peritoneal cells (table I). This radioactivity, however, accounts for less than 10 per cent. of the total 51Cr with more than 89 per cent. being TABLEI The distribution of radioactivity in I , 5 and 9 hr sponges from rats injected intravenously with sodium caseinate elicited peritoneal PMNs Time hrs. 1 5 9
No. of
animals 10 10 10
Total
c.p.m.1
sponge
103.212275 162.1 h19.7 237.01t25.5
Percentage of total c.p.m. A
Cell debris 0.1 0.9 1.2
Cells 1.1 4.9
9.4
Supernatant 98.8
,
94.2 89.4
found as free 51Cr in the supernatant fraction. It was calculated that 1.2 x 105 exudate cells/ml migrated into 5 hr sponges. A similar distribution of 51Cr was found in 5 hr sponge exudates following the intravenous injection of labelled exudate cells elicited by either starch or glycogen. The intravenous injection of reduced 51Cr in Medium 199, in an amount equivalent to the steady state level of plasma radioactivity in rats injected intravenously with labelled peritoneal exudate cells, was used to determine the proportion of the free 51Cr in the supernatant fraction of sponge exudates which could be accounted for by 51Cr free in the plasma (see Perper e f al.). Table I1 shows the c.p.m. in the supernatant of sponge exudates expressed as equivalent
18
CUNNINGHAM, SMITH, FORD-HUTCHINSON AND WALKER
TABLE I1 Radiochromium in supernatants of sponge exudates expressed as equivalent mls of plasma Time of sponge implantation
(W 1 5 9
,
Source of 5lCr
Reduced 5lCr solution (i.v.) 0.07 0.15 022
L
Labelled peritoneal cells (i.v.) 0.06 0.13 0.23
mls of plasma. These results suggest that after intravenous injection of 51Cr labelled cells or reduced 51Cr all the radioactivity in the supernatant fraction could be accounted for by plasma leakage. No significant counts were found in the circulating cells after the injection of reduced 51Cr. EfSect of peritoneal cells on accumulation of leucocytes into 5 hr sponge exudates Intravenous adminstration of peritoneal exudate cells produce a significant reduction in the number of cells accumulating in 5 hr sponge exudates. This effect was seen in both the outbred Wistar rats used throughout the work and in the two strains of inbred rats (table 111). TABLEI11 Leucocyte accumulation in 5 hr sponge exudates Rat strain n Control Test 6.9f0.1* Outbred Wistar/OLAC 10 21.0*2.5 5 8.4f0.06 4.0f0.05* Inbred WAGlOLAC 0.8f0.06* Inbred F344/OLAC 5 3.6f002 Results expressed as means *S.E. x 105/m1. Control rats injected with 1 ml Medium 199; test rats injected with sodium caseinate-induced peritoneal exudate cells. * P < 0.005 compared with corresponding control.
Chemotaxis in vitro Peripheral, peritoneal and labelled peritoneal cells showed a significant chemotactic response to serum generated chemotactic factors in vitro. The peritoneal cell response was significantly less than that of the peripheral cells (table IV).
DISCUSSION Peritoneal exudate cells labelled with 51Cr have been used for the study of chemotaxis in vitro (Gallin, Clark and Kimball, 1973) and the accumulation of leucocytes at inflammatory sites in vivo (Perper et al.; Jones, Richardson and Kay, 1977). In experiments utilising 51Cr labelled peritoneal cells for in-vivo studies it has been assumed, first, that the radioactivity accumulating at the inflammatory site is accounted for by the presence of labelled cells that have emigrated out of the vasculature into the inflammatory exudate and secondly, that the peritoneal cells behave in an identical fashion to the circulating cell population.
POLYMORPH MIGRA TION
19
TABLE IV Comparison of the in-vitro chemotaxis of peripheral and sodium caseinate induced peritoneal neutrophils Cell source n Directed migration? 15.3f2.3 Peripheral 8 Peritoneal 8 7.7*0.88* 7.2+0.67* Labelled peritoneal 8 Results expressed as means +S.E. * P
