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Quantitation of neutrophil infiltration in vivo
Immunology Letters, 1 (1979) 27-30
© Elsevier/North-HollandBiomedicalPress
Q U A N T I T A T I O N O F N E U T R O P H I L I N F I L T R A T I O N IN VIVO Andrew C. ISSEKUTZ and Henry Z. MOVAT Division o f Experimental Pathology, Department of Pathology, University of Toronto, Medical Sciences Building, Toronto, Ont., Canada M5S 1A8
(Accepted 17 March 1979)
1. Summary
3. Materials and methods
Neutrophils accumulate at sites of inflammation and infection. This report describes a method for quantitative neutrophil accumulation at such sites in vivo in rabbits by using chromium-51 labelled rabbit blood neutrophils.
3.1. L e u k o c y t e labelling
2. Introduction The migration or chemotaxis of neutrophils have been studied extensively in vitro. In contrast, far fewer reports have tried to examine this function of neutrophils under in vivo conditions. Perper et al. [1] and Jones et al. [2] used adoptively transferred [SlCr]-labelled neutrophils in the rat and guinea pig respectively, to quantitate neutrophil localization in inflammation or in response to injected chemotactic factors. In both of these studies the accumulation of [SlCr]-labelled leukocytes in response to a chemotactic or inflammatory stimulus was only 3-4-fold greater than in control or uninflamed sites. This relatively poor specific localization of labelled neutrophils may have been related to the use, by both authors of peritoneal exudate neutrophils. These cells are known to respond to chemotactic factors less vigorously in vitro than blood neutrophils [3], perhaps because peritoneal exudate neutrophils have already been stimulated to migrate out of the blood. For this reason it is desirable to use labelled blood neutrophils to study neutrophil migration in vivo. This report describes a method in which this was done.
All solutions were made up in pyrogen-free water (Baxter Travenol, Malton, Ont., Canada) and sterile plasticware was used. From the central ear artery of New-Zealand albino rabbits (2.5-3.0 kg), 30 ml of blood was collected in 0.2% ethylene diaminetetraacetic acid (EDTA) and 5 ml of blood in heparin (10 U/ml). From the heparinized blood, platelet poor plasma was obtained by two successive centrifugations (400g for 10 min and 3000g for 30 min). Leukocyte-rich plasma (LRP) was obtained from the EDTA anticoagulated blood by mixing 1 vol of 1% hydroxyethyl cellulose, (Polysciences, Warrington, Pa., USA) with 4 vol. of blood and incubating at 37°C. After 3 0 - 4 5 min the LRP was removed and centrifuged at 250 g for 10 min at 4°C. The leukocytes, with contaminating red cells, were resuspended at a concentration of 5 × 107 WBC/ml in Ca 2÷, Mg2÷free Tyrode's solution containing 5% heparinized autologous plasma and 100/aCi of Na2 [51Cr]04 (New England Nuclear, Lachine, Que., Canada). This suspension was incubated at 37°C for 30 rain. The labelled cells were washed once in 12 vol. of Ca 2÷, Mg2+-free Tyrode's solution (250 g for 10 min), resuspended in 5 ml of the same buffer and infused intravenously into the marginal ear vein of the original blood-donating rabbit. In some experiments the neutrophils were separated from mononuclear cells by Percoll (Pharmacia, Dorval, Que., Canada) density gradient centrifugation. For this the LRP was diluted with an equal volume of 27
pyrogen-free saline and 20 ml was layered onto 10 ml of Percoll, density 1.084 g/ml in 29 × 113 mm (50 ml) tubes (Falcon No. 2070, Oxnard, CA). The cells were centrifuged at 600 g for 20 rain at 4°C. The pellet was washed once in Ca 2., Mg2+-free Tyrode's solution and then labelled with Na2[SlCr]04 as described above. 3.2. Inflammatory stimuli The complement system in heparinized rabbit plasma was activated by incubating it with zymosan (5 mg/ml; Sigma, St. Louis, MO) for 60 min at 37°C. The zymosan was removed by centrifugation. Eschericia coli 055:B5 was grown overnight in nutrient broth (Difco, Detroit, MI), washed twice in 0.9% saline and the concentration of bacteria was estimated spectrophotometrically. 3.3. Skin lesion Within 30 min of infusing the labelled leukocytes, 0.15 ml of a control or inflammatory stimulus was injected in quadruplicate intradermally into 3 0 - 3 6 sites on the backs of rabbits. After 4 h, the rabbits were killed, the skin of the back was removed and blood in the large cutaneous veins was manually expressed towards the margins. The skin was frozen, the skin sites were punched out with a 15 mm corkborer and the amount of radioactivity in the sites was counted in an Intertechnique gamma counter at a setting of 7 0 - 4 0 0 Kev.
4. Results and discussion From 30 ml EDTA anticoagulated rabbit blood, approximately 180 × 106 leukocytes (range: 80 X 106 to 260 X 106) and 70 X 106 neutrophils (range: 40 × 10 6 t o 110 X 106) were recovered. In two of these experiments the leukocytes were fractionated on Percoll density gradients in order to remove mononuclear cells and platelets prior to labelling with [SlCr]. Although the leukocytes in this preparation were greater than 95% neutrophils, contaminating red cells were still present ( 2 - 3 per neutrophil). Any attempt to lyse these red ceils adversely affected the in vivo circulation and localization of the labelled neutrophils. Therefore, red cell lysis was not employed, The results with this neutro28
phil preparation were comparable to those obtained when the mixed leukocyte population was labelled (see below). For this reason, and to expedite leukocyte processing, in many experiments all of the leukocytes were labelled. On the average 1.6 × 10 6 cpm were infused (range: 1 × l 0 6 t o 2.3 X 10 6 cpm). In two experiments in which the mixed leukocytes were labelled, Percoll density gradients were used to separate an aliquot of the infused leukocyte radioactivity and NHaCI was used to lyse red blood cells. Thirty-five percent of the radioactivity was on red cells. Of the leukocyte radioactivity, 35% (mean 4.4 X l0 s cpm) was on mononuclear cel!s (spec. act. 10 cpm/103 cells). The remaining 65% (8.4 X 10 s cpm) was on neutrophils, which labelled with a higher specific activity (22 cpm/103 cells) than the mononuclear cells. Platelet radioactivity was minimal. In two experiments blood samples were collected at various times to determine the distribution of radioactivity among the cells in the circulation and to calculate the neutrophil disappearance rate. One hour after injection, 1 ml of blood contained 70 cpm in plasma, 330 cpm on mononuclear cells, 850 cpm on neutrophils and 4000 cpm on red cells. The half-life of labelled neutrophils in the circulation averaged 3.6 h (range: 3.2-4.0) and 29-33% of the neutrophils were circulating. These results with in vitro [SlCr]labelled neutrophils are nearly identical to the results of Price and Dale [4], who labelled rabbit neutrophils in vivo with [3H]thymidine. They also found that the half-life of rabbit neutrophils was very short (3.2 h) and that only 30% of these cells circulate at any one time. The remaining neutrophils are thought to be in the marginating pool. Figure 1 shows the effect of injecting skin sites with various concentrations of live E. eoli 055:B5. A logarithmic relationship was found between the number of viable bacteria injected and the accumulation of [SICr]-labelled neutrophils. As few as 103 bacteria caused measurable neutrophil localization (341 -+ 40 S.E.M.; n = 3; saline = 65 -+ 9) and 2 X 107 bacteria induced a maximal response (3910 cpm + 220). In these experiments histological sections confirmed that greater than 95% of the infiltrating cells were neutrophils (fig.2). Activation of complement by zymosan is known to generate C5a, which is a potent chemotactic factor for neutrophils [5]. Zymosan-activated plasma (ZAP)
A C C U M U L A T I O N OF L E U K O C Y T E S IN S K I N L E S I O N S I N D U C E D BY E. coli 10 4
10:
E C3 10 2
I
I
I
t
I
1
I
I
I
101 10 2 10 3 10 4 10 5 10 ~ 10 7 10 s 10 9 N u m b e r of E. coil Injected
Fig.1. The effect of injecting E. coli 055 :B5 on [ SlCr]qabelled neutrophil accumulation. The circulating leukocytes of rabbits were labelled by the i.v. infusion of autologous [SlCr]labelled leukocytes (2 experiments) or purified neutrophils (1 experiment). Thirty min later varying numbers of E. coil were injected intradermally. Four hours later the rabbits were killed and the radioactivity in the skin sites was counted. Points are means +_S.E.M.
also induced a marked accumulation of [SlCr]-labelled neutrophils (confirmed histologically and by employing purified neutrophils) (ZAP = 2560 • 148; unactivated plasma = 54 -+ 7 S.E.M., n = 4). Hemorrhage is often observed during inflammation. Therefore the presence in the circulation of labelled red cells (4000 cpm/ml of blood) concerned us. However, we feel that red cell extravasation did not contribute significantly to the [SlCr] accumulation because the ZAP lesions had barely visible hemorrhage. In addition, hemorrhage in the E. coli lesions occurred mainly after 4 h [6], while the [S~Cr] and neutrophil accumulation (histologically) occurred in the first 4 h [6]. Lastly, we have recently used [SgFe]-labelled red cells to quantitate hemorrhage [6,7] in E. coli lesions. These results indicate that the maximum hemorrhage in these sites does not exceed 10-15/al of blood. This amount of red cell extravasation could account for only 4 0 - 6 0 cpm of the total of 3 5 0 0 4000 cpm of [SlCr] in E. coli lesions.
Fig.2. The morphological appearance of the infiltrating cells into four hour old E. coli skin lesions. Nearly all of the cells are polymorphonuclear leukocytes.
As mentioned above, about 35% of the radioactivity was present on mononuclear cells. These cells could have contributed to the [SlCr] accumulation in the lesions. Therefore, in two experiments we separated the leukocytes on PercoU density gradients to obtain 95% pure neutrophils (plus contaminating red cells but no platelets) and labelled these cells. The results in these experiments were comparable to the results obtained when mixed leukocytes were labelled. One of the 3 experiments in fig.1 employed these purified neutrophils. Furthermore, histology confirmed that nearly all of the infiltrating cells were neutrophils (fig.2). In conclusion, we feel that by labelling blood 29
neutrophils with [SlCr] one can measure reproducibly and with great sensitivity neutrophil migration or chemotaxis in vivo in response to a variety of Stimuli.
Acknowledgements This work was supported by the Medical Research Council of Canada Grant MT-1251. A.C.I. is a Research Fellow of the Medical Research Council of Canada, H.Z.M. is a Research Associate of the Medical Research Council of Canada. The authors wish to thank Marica Mikhail for her expert secretarial assistance.
30
References [ 1 ] Perper, R. J., Sanda, M., Chinea, G. and Oronsky, A. L. (1974) J. Lab. Clin. Med. 84,378-393. [2] Jones, D. G., Richardson, D. L. and Kay, A. B. (1977) Br. J. Haematol. 35, 19-24. [3] Ward, P. A. and Becker, E. L. (1970) J. Immunol. 105, 1057-1067. [4] Price, T. H. and Dale, D. C. (1977) J. Clin. Invest. 59, 475 -480. [5] Fernandez, H. N., Henson, P. M., Otani, A., and Hugli, T. E. (1978) J. Immunol. 120,109-115. [6] Kopaniak, M. M., Issekutz, A. C. and Movat, H. Z. (1979) Fed. Proc. 38, (in press) (Abstract). [7] Kopaniak, M. M., Issekutz, A. C. and Movat, H. Z. submitted for publication.
© Elsevier/North-HollandBiomedicalPress
Q U A N T I T A T I O N O F N E U T R O P H I L I N F I L T R A T I O N IN VIVO Andrew C. ISSEKUTZ and Henry Z. MOVAT Division o f Experimental Pathology, Department of Pathology, University of Toronto, Medical Sciences Building, Toronto, Ont., Canada M5S 1A8
(Accepted 17 March 1979)
1. Summary
3. Materials and methods
Neutrophils accumulate at sites of inflammation and infection. This report describes a method for quantitative neutrophil accumulation at such sites in vivo in rabbits by using chromium-51 labelled rabbit blood neutrophils.
3.1. L e u k o c y t e labelling
2. Introduction The migration or chemotaxis of neutrophils have been studied extensively in vitro. In contrast, far fewer reports have tried to examine this function of neutrophils under in vivo conditions. Perper et al. [1] and Jones et al. [2] used adoptively transferred [SlCr]-labelled neutrophils in the rat and guinea pig respectively, to quantitate neutrophil localization in inflammation or in response to injected chemotactic factors. In both of these studies the accumulation of [SlCr]-labelled leukocytes in response to a chemotactic or inflammatory stimulus was only 3-4-fold greater than in control or uninflamed sites. This relatively poor specific localization of labelled neutrophils may have been related to the use, by both authors of peritoneal exudate neutrophils. These cells are known to respond to chemotactic factors less vigorously in vitro than blood neutrophils [3], perhaps because peritoneal exudate neutrophils have already been stimulated to migrate out of the blood. For this reason it is desirable to use labelled blood neutrophils to study neutrophil migration in vivo. This report describes a method in which this was done.
All solutions were made up in pyrogen-free water (Baxter Travenol, Malton, Ont., Canada) and sterile plasticware was used. From the central ear artery of New-Zealand albino rabbits (2.5-3.0 kg), 30 ml of blood was collected in 0.2% ethylene diaminetetraacetic acid (EDTA) and 5 ml of blood in heparin (10 U/ml). From the heparinized blood, platelet poor plasma was obtained by two successive centrifugations (400g for 10 min and 3000g for 30 min). Leukocyte-rich plasma (LRP) was obtained from the EDTA anticoagulated blood by mixing 1 vol of 1% hydroxyethyl cellulose, (Polysciences, Warrington, Pa., USA) with 4 vol. of blood and incubating at 37°C. After 3 0 - 4 5 min the LRP was removed and centrifuged at 250 g for 10 min at 4°C. The leukocytes, with contaminating red cells, were resuspended at a concentration of 5 × 107 WBC/ml in Ca 2÷, Mg2÷free Tyrode's solution containing 5% heparinized autologous plasma and 100/aCi of Na2 [51Cr]04 (New England Nuclear, Lachine, Que., Canada). This suspension was incubated at 37°C for 30 rain. The labelled cells were washed once in 12 vol. of Ca 2÷, Mg2+-free Tyrode's solution (250 g for 10 min), resuspended in 5 ml of the same buffer and infused intravenously into the marginal ear vein of the original blood-donating rabbit. In some experiments the neutrophils were separated from mononuclear cells by Percoll (Pharmacia, Dorval, Que., Canada) density gradient centrifugation. For this the LRP was diluted with an equal volume of 27
pyrogen-free saline and 20 ml was layered onto 10 ml of Percoll, density 1.084 g/ml in 29 × 113 mm (50 ml) tubes (Falcon No. 2070, Oxnard, CA). The cells were centrifuged at 600 g for 20 rain at 4°C. The pellet was washed once in Ca 2., Mg2+-free Tyrode's solution and then labelled with Na2[SlCr]04 as described above. 3.2. Inflammatory stimuli The complement system in heparinized rabbit plasma was activated by incubating it with zymosan (5 mg/ml; Sigma, St. Louis, MO) for 60 min at 37°C. The zymosan was removed by centrifugation. Eschericia coli 055:B5 was grown overnight in nutrient broth (Difco, Detroit, MI), washed twice in 0.9% saline and the concentration of bacteria was estimated spectrophotometrically. 3.3. Skin lesion Within 30 min of infusing the labelled leukocytes, 0.15 ml of a control or inflammatory stimulus was injected in quadruplicate intradermally into 3 0 - 3 6 sites on the backs of rabbits. After 4 h, the rabbits were killed, the skin of the back was removed and blood in the large cutaneous veins was manually expressed towards the margins. The skin was frozen, the skin sites were punched out with a 15 mm corkborer and the amount of radioactivity in the sites was counted in an Intertechnique gamma counter at a setting of 7 0 - 4 0 0 Kev.
4. Results and discussion From 30 ml EDTA anticoagulated rabbit blood, approximately 180 × 106 leukocytes (range: 80 X 106 to 260 X 106) and 70 X 106 neutrophils (range: 40 × 10 6 t o 110 X 106) were recovered. In two of these experiments the leukocytes were fractionated on Percoll density gradients in order to remove mononuclear cells and platelets prior to labelling with [SlCr]. Although the leukocytes in this preparation were greater than 95% neutrophils, contaminating red cells were still present ( 2 - 3 per neutrophil). Any attempt to lyse these red ceils adversely affected the in vivo circulation and localization of the labelled neutrophils. Therefore, red cell lysis was not employed, The results with this neutro28
phil preparation were comparable to those obtained when the mixed leukocyte population was labelled (see below). For this reason, and to expedite leukocyte processing, in many experiments all of the leukocytes were labelled. On the average 1.6 × 10 6 cpm were infused (range: 1 × l 0 6 t o 2.3 X 10 6 cpm). In two experiments in which the mixed leukocytes were labelled, Percoll density gradients were used to separate an aliquot of the infused leukocyte radioactivity and NHaCI was used to lyse red blood cells. Thirty-five percent of the radioactivity was on red cells. Of the leukocyte radioactivity, 35% (mean 4.4 X l0 s cpm) was on mononuclear cel!s (spec. act. 10 cpm/103 cells). The remaining 65% (8.4 X 10 s cpm) was on neutrophils, which labelled with a higher specific activity (22 cpm/103 cells) than the mononuclear cells. Platelet radioactivity was minimal. In two experiments blood samples were collected at various times to determine the distribution of radioactivity among the cells in the circulation and to calculate the neutrophil disappearance rate. One hour after injection, 1 ml of blood contained 70 cpm in plasma, 330 cpm on mononuclear cells, 850 cpm on neutrophils and 4000 cpm on red cells. The half-life of labelled neutrophils in the circulation averaged 3.6 h (range: 3.2-4.0) and 29-33% of the neutrophils were circulating. These results with in vitro [SlCr]labelled neutrophils are nearly identical to the results of Price and Dale [4], who labelled rabbit neutrophils in vivo with [3H]thymidine. They also found that the half-life of rabbit neutrophils was very short (3.2 h) and that only 30% of these cells circulate at any one time. The remaining neutrophils are thought to be in the marginating pool. Figure 1 shows the effect of injecting skin sites with various concentrations of live E. eoli 055:B5. A logarithmic relationship was found between the number of viable bacteria injected and the accumulation of [SICr]-labelled neutrophils. As few as 103 bacteria caused measurable neutrophil localization (341 -+ 40 S.E.M.; n = 3; saline = 65 -+ 9) and 2 X 107 bacteria induced a maximal response (3910 cpm + 220). In these experiments histological sections confirmed that greater than 95% of the infiltrating cells were neutrophils (fig.2). Activation of complement by zymosan is known to generate C5a, which is a potent chemotactic factor for neutrophils [5]. Zymosan-activated plasma (ZAP)
A C C U M U L A T I O N OF L E U K O C Y T E S IN S K I N L E S I O N S I N D U C E D BY E. coli 10 4
10:
E C3 10 2
I
I
I
t
I
1
I
I
I
101 10 2 10 3 10 4 10 5 10 ~ 10 7 10 s 10 9 N u m b e r of E. coil Injected
Fig.1. The effect of injecting E. coli 055 :B5 on [ SlCr]qabelled neutrophil accumulation. The circulating leukocytes of rabbits were labelled by the i.v. infusion of autologous [SlCr]labelled leukocytes (2 experiments) or purified neutrophils (1 experiment). Thirty min later varying numbers of E. coil were injected intradermally. Four hours later the rabbits were killed and the radioactivity in the skin sites was counted. Points are means +_S.E.M.
also induced a marked accumulation of [SlCr]-labelled neutrophils (confirmed histologically and by employing purified neutrophils) (ZAP = 2560 • 148; unactivated plasma = 54 -+ 7 S.E.M., n = 4). Hemorrhage is often observed during inflammation. Therefore the presence in the circulation of labelled red cells (4000 cpm/ml of blood) concerned us. However, we feel that red cell extravasation did not contribute significantly to the [SlCr] accumulation because the ZAP lesions had barely visible hemorrhage. In addition, hemorrhage in the E. coli lesions occurred mainly after 4 h [6], while the [S~Cr] and neutrophil accumulation (histologically) occurred in the first 4 h [6]. Lastly, we have recently used [SgFe]-labelled red cells to quantitate hemorrhage [6,7] in E. coli lesions. These results indicate that the maximum hemorrhage in these sites does not exceed 10-15/al of blood. This amount of red cell extravasation could account for only 4 0 - 6 0 cpm of the total of 3 5 0 0 4000 cpm of [SlCr] in E. coli lesions.
Fig.2. The morphological appearance of the infiltrating cells into four hour old E. coli skin lesions. Nearly all of the cells are polymorphonuclear leukocytes.
As mentioned above, about 35% of the radioactivity was present on mononuclear cells. These cells could have contributed to the [SlCr] accumulation in the lesions. Therefore, in two experiments we separated the leukocytes on PercoU density gradients to obtain 95% pure neutrophils (plus contaminating red cells but no platelets) and labelled these cells. The results in these experiments were comparable to the results obtained when mixed leukocytes were labelled. One of the 3 experiments in fig.1 employed these purified neutrophils. Furthermore, histology confirmed that nearly all of the infiltrating cells were neutrophils (fig.2). In conclusion, we feel that by labelling blood 29
neutrophils with [SlCr] one can measure reproducibly and with great sensitivity neutrophil migration or chemotaxis in vivo in response to a variety of Stimuli.
Acknowledgements This work was supported by the Medical Research Council of Canada Grant MT-1251. A.C.I. is a Research Fellow of the Medical Research Council of Canada, H.Z.M. is a Research Associate of the Medical Research Council of Canada. The authors wish to thank Marica Mikhail for her expert secretarial assistance.
30
References [ 1 ] Perper, R. J., Sanda, M., Chinea, G. and Oronsky, A. L. (1974) J. Lab. Clin. Med. 84,378-393. [2] Jones, D. G., Richardson, D. L. and Kay, A. B. (1977) Br. J. Haematol. 35, 19-24. [3] Ward, P. A. and Becker, E. L. (1970) J. Immunol. 105, 1057-1067. [4] Price, T. H. and Dale, D. C. (1977) J. Clin. Invest. 59, 475 -480. [5] Fernandez, H. N., Henson, P. M., Otani, A., and Hugli, T. E. (1978) J. Immunol. 120,109-115. [6] Kopaniak, M. M., Issekutz, A. C. and Movat, H. Z. (1979) Fed. Proc. 38, (in press) (Abstract). [7] Kopaniak, M. M., Issekutz, A. C. and Movat, H. Z. submitted for publication.