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Immaturity of the inflammatory response of the chick chorioallantoic membrane
Toxic. in Vitro Vol. 4, No. 4/5, pp. 324-326, 1990 Printed in Great Britain. All rights reserved
0887-2333/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
I M M A T U R I T Y OF THE I N F L A M M A T O R Y RESPONSE OF THE CHICK CHORIOALLANTOIC M E M B R A N E J. V. FRIEND,R. W. R. CREVEL,T. C. WILLIAMS and W. E. PARISH Unilever Research & Engineering, Environmental Safety Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK Al~tract--Previous tests on the chick embryo chorioallantoic membrane (CAM) had shown the 24- to 48-hr response to irritants to be necrosis, with a primitive granulation proliferation at the periphery. There were few heterophils (avian neutrophils). The present investigation confirmed the immaturity of blood leucocyte development. Few heterophils were seen at 14 days but were found in significant numbers on the 19th and 20th days just before hatching. The numbers of blood heterophils in 14-day-old embryos were almost doubled and the number of immature granulocytes slightly reduced when the CAM was treated with zymosan or N-formylmethionyl peptide, which are chemo-attractants for mammalian neutrophils. Treatment with chemicals that do not have selective activity for neutrophils, for example a surfactant or alcohol, did not stimulate this change. It was not feasible to pretreat 14-day-old embryos to increase the number of heterophils or to use 19- to 21-day-old embryos as a model to detect irritants inducing an acute inflammatory response, or to make the CAM more relevant as a substitute for the in vivo eye irritation test. No difference was found in the phagocytic ability of macrophages of 14-day-old embryos and 16-wk-old adults. Macrophages from both sources contained lactate dehydrogenase and fl-glucuronidase revealed by histochemical and fluorometric examination, and non-specific esterase by histochemistry, though staining was stronger in cells from the adults.
was taken in a heparin-treated syringe from the vessels linking the embryo to the C A M , and diluted in Eagle's M i n i m u m Essential Medium ( E M E M ) supplemented with 10% chicken serum, and small drops were placed on microscope slides. To drops in duplicate were added carmine, latex or starch particles, and the slides were incubated at 37°C in a moist chamber containing 5% CO2 in air for 4 or 17 hr. The cells were then rinsed, fixed and stained for examination. After the blood was taken, the embryo was removed from the egg and killed. The peritoneal cavity was rinsed with E M E M and drops containing macrophages were placed on microscope slides and treated as above. The C A M , retina, heart, liver and spleen of the embryo were also removed. They were either chopped into small pieces or treated overnight with cold trypsin. The chopped pieces, on coverglasses in Leighton Tubes containing E M E M and 10% chicken serum, were treated with the particles immediately, or after 6 or 24 hr in culture. The trypsinized cell suspensions from the same organs were treated in the same manner as the fragments. Macrophages from the peritoneum of freshly killed adult chickens were prepared as for the embryo cells, providing a control. Suspensions of macrophages from the embryos and adult chickens were used to make cytospin slides, and these and cultures of attached cells were stained for the presence of lactate dehydrogenase, reduced nicotinamide adenine dinucleotide phosphate ( N A D P H ) , non-specific esterase, acid phosphatase and fl-glucuronidase. Mouse peritoneal macrophages were used as a control.
Introduction The chick chorioallantoic membrane (CAM), being a connective tissue sheet with a visible blood supply, has been proposed as a substrate to identify the irritant potential of substances, particularly for the eye. The tests proposed are of two types: (a) rapid, within minutes, changes in the blood vessels (Luepke, 1985; Luepke and Kemper, 1986); and (b) tissue degeneration and necrosis occurring in 1 or 2 days (Leighton et al., 1985). The latter type of necrosis was found to be unsuitable as a predictive test for eye irritancy (Lawrence et al., 1986; Parish, 1985; Price et al., 1986). The histological features of the site of application of the test substance were necrosis of varying depth according to severity, quickly followed at the periphery by activation of macrophages and proliferation of fibroblasts as a primitive form of granulation tissue. Heterophils (chick neutrophils) were scarce or absent so the characteristic acute inflammatory response to irritants was lacking (Parish, 1985). Further studies on the macrophages and heterophils in the response of the C A M to irritants were made to test the possibility of modifying the procedure to make it more relevant to in vivo responsiveness.
Materials and Methods T e s t s on m a c r o p h a g e s . Samples of blood and tissue were obtained from 14-day-old chick embryos. Blood Abbreviations :
CAM = chorioallantoic membrane; EMEM = Eagle's Minimum Essential Medium; NADPH =nicotinamide adenine dinucleotide phosphate. 324
Inflammatory response in CAM The lactate dehydrogenase and fl-glucuronidase content in suspensions of the above cells was also examined fluorometrically. Tests on heterophils. Differential leucocyte counts were made on blood from an allantoic vessel of chick embryos each day from age 14 to 20 days. All blood films were stained by the Leishman procedure. In order to detect any mobilization of heterophils in response to stimulation or irritation, the CAM of embryos 14/15 days old was treated with 50/zl of one of the following: 50% Freund's adjuvant with water, 2.5mg zymosan/10ml, 0.1% or 1.0% sodium lauryl sulphate, 50% ethanol, and the short-chain peptide mammalian neutrophil-activating agent 10 #g/ml N-formyl, L-methionyl, L-leucyl, L-phenylalanine in 0.5% alcohol (Schiffman et al., 1975) and 0.5% alcohol as the control for that reagent. To test for the effect of trauma the CAM was stroked 10-20 times with a spatula. Differential blood counts from an allantoic vessel sample were made 4 hr after treatment. Results Tests on macrophages
The macrophages of the embryo were actively phagocytic and of equal ability to those of the adult bird. All sources of embryo cells, namely blood, peritoneal washings, CAM and tissues contained phagocytic cells, though they were less numerous in the blood preparations. Histochemical staining showed reaction products for lactate dehydrogenase, non-specific esterase, acid phosphatase and/~-glucuronidase; no NADPH was detected. Each enzyme was present in cells stained immediately on isolation, and after 4 hr and overnight incubation. There were no macrophages in the embryo blood films. There were essentially no differences in staining reactions between the embryo and adult cells, though the embryo cells stained more weakly and were fewer in number. The fluorometric analysis showed all cell suspensions to contain lactate dehydrogenase and fl-glucuronidase, thus confirming the histochemical findings.
325
Tests on heterophils
Differential leucocyte counts on films of embryo blood showed few cells and it was sometimes difficult to find 100 cells at 14-18 days. In three tests on 14-day-old embryos an average total of 46 leucocytes was seen in 15 embryos, composed of an average of 25 heterophils and 20 immature granulocytes. No monocytes were seen at 14 days. There was also considerable variation in size of erythrocytes and their nuclei, and the cytoplasm tended to be basophilic, though at 17 days the erythrocytes more resembled those of the adult in morphology and staining. In the 19- and 20-day-old embryos leucocytes and thrombocytes were numerous. Of 100 cells, about 90 were heterophils, 2 were monocytes, 4 eosinophils, 1 basophil and 3 immature granulocytes. To compare these findings with those of adult vessels 6 chickens 16 wk old had a mean total white blood cell count of 11.95 × 109/litre, of which 32.8% were heterophils, 51.2% lymphocytes, 5.7% monocytes, 5.5% eosinophils and 4.8% basophils. Stimulation of the CAM of 14-day-old embryos by the reagents stated above showed some inconsistent decrease in the number of immature granulocytes compared with the untreated controls (Table 1). The decrease in immature forms was less with stimuli that activate mammalian neutrophils (zymosan, N-formyl compound and trauma) than following treatment of the CAM with Freund's adjuvant and chemicals. Treatment with the two potent activators of mammalian neutrophils also nearly doubled the number of blood heterophils compared with the untreated controls (Table 1). Discussion In a previous study it was reported that the histological examination of the 14-day-old CAM treated with irritants for 24-48 hr showed necrosis at the site, quickly followed on the periphery with primitive granulation tissue, comprising activated macrophages and proliferating fibroblasts. There were few or no heterophils (Parish, 1985). The paucity of these neutrophil equivalents was considered to be the reason for the absence of an acute inflammatory response as seen in adult chickens or mammals.
Table 1. Averageblood leucocytedifferentialcountsof 14-day-oldchick embryos,after stimulation of the CAM with 50gl reagent,and blood sampletaken at 4 hr* No. of Total Immature Treatment of CAM eggs leucocytes Heterophils granulocytes 50% Freund'sadj. 6 17.8 8 9.5 Zymosan 6 58.3 45.3 12.5 0.1% SLS 6 19.3 17.6 0.6 1.0% SLS 6 30.6 28.3 1.6 50% Ethanol 6 38.6 31.6 6.1 N-Formylt 6 58.8 42.8 15.1 0.5% Alcohol 2 22.3 20.5 1.6 (solventcontrol fort) Traumas 6 42.3 28 13.1 Untreated 15 46.4 25.4 20.6 SLS = sodium lauryl sulphate *The slight discrepanciesin numbersare due to the occasional eosinophils and lymphocytesnot presented in the Table. tN-formyl, L-methionyl,L-leucyl,L-phenylalanine. :~10to 20 strokes with spatula.
326
J. V. FRIEND et al.
The data reported in this further investigation confirm that there are few heterophils in the blood of 14-day-old chick embryos, but the number suddenly increases on the 19th day, just before hatching. At this time there are very few blood monocytes or lymphocytes. When the CAM was treated with zymosan or N-formylmethionyl peptide (Schiffman et al., 1975), which are chemotactic for mammalian neutrophils, the number of heterophils in the blood of 14-day-old chick embryos was nearly twice that of the untreated controls, and the number of immature granulocytes showed a smaller reduction than in embryos treated with other chemicals. This indicates that there is a reserve of heterophiis in the 14-day-old chick embryo that may be mobilized on appropriate stimulation. Treatment with 0.1% and 1.0% sodium lauryl sulphate did not significantly increase the number of blood heterophils, and much reduced the number of immature granulocytes. This is consistent with the absence of heterophils in the histological preparations of treated CAMs in the previous study. Apart from further examination of the absence of an acute inflammatory response in the CAM, an objective of this study was to investigate the possibility of modifying the CAM to develop a heterophil (neutrophil) response. This is not feasible. Either the CAM has to be treated just before hatching, or it would have to be stimulated by agents, for example zymosan, that may mask the effect of the test substance.
Some properties of the macrophage, as the active inflammatory cell of early embryos, were compared with those of macrophages of adult birds. No difference in phagocytic activity was seen between macrophages of embryos and 16-wk-old chickens, and though cells of the embryos showed weaker staining, there was no difference in the presence of the enzymes detected histochemically. REFERENCES
Lawrence R. S., Groom M. H., Ackroyd D. M. and Parish W. E. (1986) The chorioallantoic membrane in irritation testing. Fd Chem. Toxic. 23, 497-502. Leighton J., Nassauer J. and Tchao R. (1985) The chick embryo in toxicology: an alternative to the rabbit eye. Fd Chem. Toxic. 23, 293-298. Luepke N. P. (1985) Hen's egg chorioallantoic membrane test for irritation potential. Fd Chem. Toxic. 23, 287-291. Luepke N. P. and Kemper F. H. (1986) The HET-CAM test: an alternative to the Draize eye test. Fd Chem. Toxic. 24, 495-496. Parish W. E. (1985) Ability of in vitro (corneal injury---eye organ--and chorioallantoic membrane) tests to represent histopathological features of acute eye inflammation. Fd Chem. Toxic. 23, 215-227. Price J. B., Barry M. P. and Andrews I. J. (1986) The use of the chick chorioallantoic membrane to predict eye irritants. Fd Chem. Toxic. 24, 503-505. Schiffman E., Corcoran B. A. and Wahl S. M. (1975) N-formylmethionyl peptides are chemoattractants for leucocytes. Proc. natn. Acad. Sci. U.S.A. 72, 1059-1062.
0887-2333/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
I M M A T U R I T Y OF THE I N F L A M M A T O R Y RESPONSE OF THE CHICK CHORIOALLANTOIC M E M B R A N E J. V. FRIEND,R. W. R. CREVEL,T. C. WILLIAMS and W. E. PARISH Unilever Research & Engineering, Environmental Safety Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK Al~tract--Previous tests on the chick embryo chorioallantoic membrane (CAM) had shown the 24- to 48-hr response to irritants to be necrosis, with a primitive granulation proliferation at the periphery. There were few heterophils (avian neutrophils). The present investigation confirmed the immaturity of blood leucocyte development. Few heterophils were seen at 14 days but were found in significant numbers on the 19th and 20th days just before hatching. The numbers of blood heterophils in 14-day-old embryos were almost doubled and the number of immature granulocytes slightly reduced when the CAM was treated with zymosan or N-formylmethionyl peptide, which are chemo-attractants for mammalian neutrophils. Treatment with chemicals that do not have selective activity for neutrophils, for example a surfactant or alcohol, did not stimulate this change. It was not feasible to pretreat 14-day-old embryos to increase the number of heterophils or to use 19- to 21-day-old embryos as a model to detect irritants inducing an acute inflammatory response, or to make the CAM more relevant as a substitute for the in vivo eye irritation test. No difference was found in the phagocytic ability of macrophages of 14-day-old embryos and 16-wk-old adults. Macrophages from both sources contained lactate dehydrogenase and fl-glucuronidase revealed by histochemical and fluorometric examination, and non-specific esterase by histochemistry, though staining was stronger in cells from the adults.
was taken in a heparin-treated syringe from the vessels linking the embryo to the C A M , and diluted in Eagle's M i n i m u m Essential Medium ( E M E M ) supplemented with 10% chicken serum, and small drops were placed on microscope slides. To drops in duplicate were added carmine, latex or starch particles, and the slides were incubated at 37°C in a moist chamber containing 5% CO2 in air for 4 or 17 hr. The cells were then rinsed, fixed and stained for examination. After the blood was taken, the embryo was removed from the egg and killed. The peritoneal cavity was rinsed with E M E M and drops containing macrophages were placed on microscope slides and treated as above. The C A M , retina, heart, liver and spleen of the embryo were also removed. They were either chopped into small pieces or treated overnight with cold trypsin. The chopped pieces, on coverglasses in Leighton Tubes containing E M E M and 10% chicken serum, were treated with the particles immediately, or after 6 or 24 hr in culture. The trypsinized cell suspensions from the same organs were treated in the same manner as the fragments. Macrophages from the peritoneum of freshly killed adult chickens were prepared as for the embryo cells, providing a control. Suspensions of macrophages from the embryos and adult chickens were used to make cytospin slides, and these and cultures of attached cells were stained for the presence of lactate dehydrogenase, reduced nicotinamide adenine dinucleotide phosphate ( N A D P H ) , non-specific esterase, acid phosphatase and fl-glucuronidase. Mouse peritoneal macrophages were used as a control.
Introduction The chick chorioallantoic membrane (CAM), being a connective tissue sheet with a visible blood supply, has been proposed as a substrate to identify the irritant potential of substances, particularly for the eye. The tests proposed are of two types: (a) rapid, within minutes, changes in the blood vessels (Luepke, 1985; Luepke and Kemper, 1986); and (b) tissue degeneration and necrosis occurring in 1 or 2 days (Leighton et al., 1985). The latter type of necrosis was found to be unsuitable as a predictive test for eye irritancy (Lawrence et al., 1986; Parish, 1985; Price et al., 1986). The histological features of the site of application of the test substance were necrosis of varying depth according to severity, quickly followed at the periphery by activation of macrophages and proliferation of fibroblasts as a primitive form of granulation tissue. Heterophils (chick neutrophils) were scarce or absent so the characteristic acute inflammatory response to irritants was lacking (Parish, 1985). Further studies on the macrophages and heterophils in the response of the C A M to irritants were made to test the possibility of modifying the procedure to make it more relevant to in vivo responsiveness.
Materials and Methods T e s t s on m a c r o p h a g e s . Samples of blood and tissue were obtained from 14-day-old chick embryos. Blood Abbreviations :
CAM = chorioallantoic membrane; EMEM = Eagle's Minimum Essential Medium; NADPH =nicotinamide adenine dinucleotide phosphate. 324
Inflammatory response in CAM The lactate dehydrogenase and fl-glucuronidase content in suspensions of the above cells was also examined fluorometrically. Tests on heterophils. Differential leucocyte counts were made on blood from an allantoic vessel of chick embryos each day from age 14 to 20 days. All blood films were stained by the Leishman procedure. In order to detect any mobilization of heterophils in response to stimulation or irritation, the CAM of embryos 14/15 days old was treated with 50/zl of one of the following: 50% Freund's adjuvant with water, 2.5mg zymosan/10ml, 0.1% or 1.0% sodium lauryl sulphate, 50% ethanol, and the short-chain peptide mammalian neutrophil-activating agent 10 #g/ml N-formyl, L-methionyl, L-leucyl, L-phenylalanine in 0.5% alcohol (Schiffman et al., 1975) and 0.5% alcohol as the control for that reagent. To test for the effect of trauma the CAM was stroked 10-20 times with a spatula. Differential blood counts from an allantoic vessel sample were made 4 hr after treatment. Results Tests on macrophages
The macrophages of the embryo were actively phagocytic and of equal ability to those of the adult bird. All sources of embryo cells, namely blood, peritoneal washings, CAM and tissues contained phagocytic cells, though they were less numerous in the blood preparations. Histochemical staining showed reaction products for lactate dehydrogenase, non-specific esterase, acid phosphatase and/~-glucuronidase; no NADPH was detected. Each enzyme was present in cells stained immediately on isolation, and after 4 hr and overnight incubation. There were no macrophages in the embryo blood films. There were essentially no differences in staining reactions between the embryo and adult cells, though the embryo cells stained more weakly and were fewer in number. The fluorometric analysis showed all cell suspensions to contain lactate dehydrogenase and fl-glucuronidase, thus confirming the histochemical findings.
325
Tests on heterophils
Differential leucocyte counts on films of embryo blood showed few cells and it was sometimes difficult to find 100 cells at 14-18 days. In three tests on 14-day-old embryos an average total of 46 leucocytes was seen in 15 embryos, composed of an average of 25 heterophils and 20 immature granulocytes. No monocytes were seen at 14 days. There was also considerable variation in size of erythrocytes and their nuclei, and the cytoplasm tended to be basophilic, though at 17 days the erythrocytes more resembled those of the adult in morphology and staining. In the 19- and 20-day-old embryos leucocytes and thrombocytes were numerous. Of 100 cells, about 90 were heterophils, 2 were monocytes, 4 eosinophils, 1 basophil and 3 immature granulocytes. To compare these findings with those of adult vessels 6 chickens 16 wk old had a mean total white blood cell count of 11.95 × 109/litre, of which 32.8% were heterophils, 51.2% lymphocytes, 5.7% monocytes, 5.5% eosinophils and 4.8% basophils. Stimulation of the CAM of 14-day-old embryos by the reagents stated above showed some inconsistent decrease in the number of immature granulocytes compared with the untreated controls (Table 1). The decrease in immature forms was less with stimuli that activate mammalian neutrophils (zymosan, N-formyl compound and trauma) than following treatment of the CAM with Freund's adjuvant and chemicals. Treatment with the two potent activators of mammalian neutrophils also nearly doubled the number of blood heterophils compared with the untreated controls (Table 1). Discussion In a previous study it was reported that the histological examination of the 14-day-old CAM treated with irritants for 24-48 hr showed necrosis at the site, quickly followed on the periphery with primitive granulation tissue, comprising activated macrophages and proliferating fibroblasts. There were few or no heterophils (Parish, 1985). The paucity of these neutrophil equivalents was considered to be the reason for the absence of an acute inflammatory response as seen in adult chickens or mammals.
Table 1. Averageblood leucocytedifferentialcountsof 14-day-oldchick embryos,after stimulation of the CAM with 50gl reagent,and blood sampletaken at 4 hr* No. of Total Immature Treatment of CAM eggs leucocytes Heterophils granulocytes 50% Freund'sadj. 6 17.8 8 9.5 Zymosan 6 58.3 45.3 12.5 0.1% SLS 6 19.3 17.6 0.6 1.0% SLS 6 30.6 28.3 1.6 50% Ethanol 6 38.6 31.6 6.1 N-Formylt 6 58.8 42.8 15.1 0.5% Alcohol 2 22.3 20.5 1.6 (solventcontrol fort) Traumas 6 42.3 28 13.1 Untreated 15 46.4 25.4 20.6 SLS = sodium lauryl sulphate *The slight discrepanciesin numbersare due to the occasional eosinophils and lymphocytesnot presented in the Table. tN-formyl, L-methionyl,L-leucyl,L-phenylalanine. :~10to 20 strokes with spatula.
326
J. V. FRIEND et al.
The data reported in this further investigation confirm that there are few heterophils in the blood of 14-day-old chick embryos, but the number suddenly increases on the 19th day, just before hatching. At this time there are very few blood monocytes or lymphocytes. When the CAM was treated with zymosan or N-formylmethionyl peptide (Schiffman et al., 1975), which are chemotactic for mammalian neutrophils, the number of heterophils in the blood of 14-day-old chick embryos was nearly twice that of the untreated controls, and the number of immature granulocytes showed a smaller reduction than in embryos treated with other chemicals. This indicates that there is a reserve of heterophiis in the 14-day-old chick embryo that may be mobilized on appropriate stimulation. Treatment with 0.1% and 1.0% sodium lauryl sulphate did not significantly increase the number of blood heterophils, and much reduced the number of immature granulocytes. This is consistent with the absence of heterophils in the histological preparations of treated CAMs in the previous study. Apart from further examination of the absence of an acute inflammatory response in the CAM, an objective of this study was to investigate the possibility of modifying the CAM to develop a heterophil (neutrophil) response. This is not feasible. Either the CAM has to be treated just before hatching, or it would have to be stimulated by agents, for example zymosan, that may mask the effect of the test substance.
Some properties of the macrophage, as the active inflammatory cell of early embryos, were compared with those of macrophages of adult birds. No difference in phagocytic activity was seen between macrophages of embryos and 16-wk-old chickens, and though cells of the embryos showed weaker staining, there was no difference in the presence of the enzymes detected histochemically. REFERENCES
Lawrence R. S., Groom M. H., Ackroyd D. M. and Parish W. E. (1986) The chorioallantoic membrane in irritation testing. Fd Chem. Toxic. 23, 497-502. Leighton J., Nassauer J. and Tchao R. (1985) The chick embryo in toxicology: an alternative to the rabbit eye. Fd Chem. Toxic. 23, 293-298. Luepke N. P. (1985) Hen's egg chorioallantoic membrane test for irritation potential. Fd Chem. Toxic. 23, 287-291. Luepke N. P. and Kemper F. H. (1986) The HET-CAM test: an alternative to the Draize eye test. Fd Chem. Toxic. 24, 495-496. Parish W. E. (1985) Ability of in vitro (corneal injury---eye organ--and chorioallantoic membrane) tests to represent histopathological features of acute eye inflammation. Fd Chem. Toxic. 23, 215-227. Price J. B., Barry M. P. and Andrews I. J. (1986) The use of the chick chorioallantoic membrane to predict eye irritants. Fd Chem. Toxic. 24, 503-505. Schiffman E., Corcoran B. A. and Wahl S. M. (1975) N-formylmethionyl peptides are chemoattractants for leucocytes. Proc. natn. Acad. Sci. U.S.A. 72, 1059-1062.