The
Journal of Pathology Vol. 123 No. 3 C Y C L I C A M P A N D T H E M E C H A N I S M O F LEUCOCYTE LYSOSOMAL ENZYME RELEASE D U R I N G A N IMMEDIATE HYPERSENSITIVITY REACTION IN VIVO
D. A. DEPORTER* Department of Experimental Pathology, St Bartholomew’s Hos&nl Medical School, London, England
I T is generally recognised that an insight into the control mechanisms involved in the release of lysosomal enzymes from inflammatory leucocytes could be of considerable benefit in the treatment of inflammatory diseases such as crystalinduced arthopathies and rheumatoid arthritis. Recent studies from several laboratories have implicated the cyclic nucleotides, adenosine 3’,5’-cyclic monophosphate (cyclic AMP) and guanosine 3’, 5’-cyclic monophosphate (cyclic GMP), in the control of lysosomal enzyme release from polymorphonuclear leucocytes (PMN’s). It has been demonstrated that the discharge of these potentially destructive acid hydrolases from isolated PMN’s in response to a variety of stimuli, including Ag-Ab complexes can be inhibited by artificially elevating PMN cyclic AMP levels or enhanced by increasing their cyclic GMP levels (Weissmann et al., 1971; Ignarro, 1974; Zurier et al., 1974). However, these results have not as yet been supported by suitable animal experiments. In an earlier study we presented evidence favouring a lack of correlation between lysosomal enzyme release and leucocyte cyclic AMP levels during crystal-induced (Ca pyrophosphate) pleurisy in rats (Deporter et al., 1976). We have now repeated these experiments using a different model of acute inflammation, namely a reverse passive Arthus reaction in the pleural cavity of rats (Yamamoto et al., 1975) and the results comprise the present report. MATERIALS AND
METHODS
Reverse passive Arthus reactions were produced in the pleural cavities of male Wistar rats (Yamamoto et al., 1975) and modified with theophylline and/or dibutyryl cyclic AMP administered as previously described with pyrophosphate-induced pleurisy (Deporter et al., 1976). Received 10 Dec. 1976; accepted 17 Jan. 1977. Division of Biological Sciences, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, MSG lG6, Canada.
* Present address: I. PATH.-VOL.
123 (1977)
129
I
D . B. DEPORTER
130
Cyclic AMP estimations were performed using the method of Brown ef al., (1971). Assays of B-glucuronidase and lactate dehydrogenase (LDH) activities were as before (Deporter ef a!., 1976). In some experiments the effect of elevating leucocyte AMP levels with theophylline and dibutyryl cyclic AMP on leucocyte cyclic GMP levels was studied. Leucocytes from control and drug-treated pleural effusions were fixed in 1 per cent. perchloric acid, sonicated for 30 s and centrifuged at 10,OOOg for 15 min. The clear supernatants were adjusted to pH 7.0 with KOH and recentrifuged. The clear supernatants from this second centrifugation were each ~ acid. Cyclic applied to a separate 1 ml column of Biorad equilibrated with 0 . 1 formic AMP was eluted with 2N formic acid while cyclic GMP was eluted with 4N formic acid.
Control
dC-amp
Both
Colchicine
FIG. 1.-Effect
of intrapleurally administered dibutyryl cyclic AMP (5 mM), intrapleurally administered dibutyryl cyclic AMP with theophylline (5 mM each) and intravenously administered colchicine (0.2 mg/kg) on leucocyte cyclic AMP concentration at 3 hr following the onset of an intrapleural reverse passive Arthus reaction. Values are the mean of 12 experiments.
Following freeze-drying of the eluates, the residues were assayed for cyclic G M P using a commercially available assay kit (Collaborative Research Inc., Waltham, Mass.) and for cyclic AMP as described above. In other animals the effects of colchicine on leucocyte cyclic AMP levels and lysosomal enzyme discharge during the reverse passive Arthus reaction were assessed. Colchicine (BDH, Poole, Dorset) was given intravenously (0.2 mg per kg, in saline 1 hr before eliciting the intrapleural reaction.
RESULTS Effect of dibutyryl cyclic AMP with and without theophylline on leucocyte cyclic AMP levels Administration of 5 mM dibutyryl cyclic AMP intrapleurally at the time of onset of the intrapleural Arthus reaction produced a 135 per cent. increase
LEUCOCYTE CYCLIC AMP
131
in leucocyte cyclic AMP concentration as compared with leucocytes from control reactions. When 5 mM dibutyryl cyclic AMP and 5 mM theophylline were given together, a 235 per cent. increase in leucocyte cyclic AMP content was observed (fig. 1). 25
20
1
10
5
dC-amp
Control
Both
FIG. 2.-Effect of dibutyryl cyclic AMP alone and in combination with theophylline (both) on the percentage B-glucuronidase activity released in the Arthus-induced pleural effusions. Values are the mean of 12 experiments. extracellular enzyme activity x 100 percentage released = cellular extracellular activity ~
+
TABLEI Efect of dibutyryl cyclic AMP with or without theophylline on extracellular L D H levels; the values represent the mem of twelve experiments Control
Percentage LDH released 14.5f2.0
dibutyryl cyclic AMP
20.0f2.9
dibutyryl cyclic AMP theophylline
183+4.9
+
Effect of dibutyryl cyclic AMP with and without theophylline on the percentage leucocyte lysosomal B-glucuronidase and leucocyte lactate dehydrogenase activities released. Administration of dibutyryl cyclic AMP produced no reduction in the amount of B-glucuronidase released into the 3-hr Arthus-induced pleural effusions. When dibutyryl cyclic AMP and theophylline were both injected intrapleurally at the time of onset of the Arthus reaction, there was a 30 per cent. increase in enzyme release (fig. 2). The LDH results were slightly different (table 1). The smallest amounts of LDH were released in control reactions
D. A . DEPORTER
132
but in all three groups studied the percentage LDH released was never more than 25 per cent. of the total leucocyte LDH content.
EfSect of administering dibutyryl cyclic AMP and theophylline on leucocyte cyclic GMP levels The results of these experiments are shown in table 11. Cyclic AMP and cyclic GMP levels were assayed for the same pleural exudates after separation TABLEI1 EfSect of 5 mM dibutyryl cyclic AMP and 5 mM theophylline on leucocyte cyclic GMP and cyclic A M P levels from the same pleural reactions; the values represent the mean of twelve experiments Cyclic GMP/lOg cells Cyclic AMP/lOs cells Control Arthus reaction
0.83 pmoles
65 pmoles
Drug-modified Arthus reaction
1.3 pmoles
247 pmoles
of the two nucleotides on mini-columns of Biorad. Whereas the treatment produced a 280 per cent. increase in leucocyte cyclic AMP concentration it produced a 57 per cent. increase in leucocyte cyclic GMP content. 200
20
1150
~
2
EL
.-
5
0%
I
I
W
tT rn
15 1100
-I
E C
g
10
Y
%
'.SO 5
FIG. 3.--Effect of colchicine on the extracellular concentration of B-glucuronidase activity (left side) and of the total extracellular B-glucuronidase activity (right side). Values are the mean of 12 experiments. Cross-hatch=colchicine; diagonal stripe=control.
Effect of colchicine on B-glucuronidase released into pleural effusions As can be seen in fig. 3, pretreatment of animals with intravenous colchicine produced no decrease in the amount of B-glucuronidase released into the Arthus-
LEUCOCYTE CYCLIC AMP
133
induced pleural effusions. In contrast to this lack of effect of colchicine on enzyme release, the same pretreatment produced a 154 per cent. increase in leucocyte cyclic AMP concentration (fig. 1). DISCUSSION
We have previously demonstrated an apparent lack of inhibition of lysosomal enzyme discharge from leucocytes following therapeutic elevation of cellular cyclic AMP levels during pyrophosphate pleurisy in rats (Deporter et al., 1977). The present results in a different model of acute inflammation, an immediate hypersensitivity reaction in the pleural cavity of rats, provide further evidence for this conclusion. Further, they tend to dispel any argument suggesting that the earlier results were due primarily to an effect of free calcium ions. It might have been argued, for example, following the work of Smith and Ignarro (1975) and others (Schultz et al., 1973), that whereas the drugs used (dibutyryl cyclic AMP and theophylline) produced a marked increase in leucocyte cyclic AMP levels, the extra free calcium might have produced a much larger increase in cyclic GMP with concomitant lysosomal enzyme discharge. Administration of dibutyryl cyclic AMP with or without theophylline into the pleural cavity of rats at the time of initiation of an immediate hypersensitivity reaction in the same site produced a 135 to 235 per cent. increase in leucocyte cyclic AMP levels but had no significant effect on secretion of the lysosomal hydrolase, B-glucuronidase. This observation that cyclic AMP does not monitor lysosomal release in vivo is in direct opposition to the in-vitro work of Weissmann’s group (e.g., Weissmann, Dukor and Zurier, 1971). The reason for this lack of agreement is not readily apparent. These other workers did use concentrations of dibutyryl cyclic AMP and theophylline approximating our own dose regimens. Furthermore, for the most part they utilised mixed populations of leucocytes rather than purified PMN’s which again corresponds well to the population of cells present in a pleural effusion. One important point of diflerence, however, is that in the in-vitro experiments the leucocytes were pre-incubated with agents designed to increase cyclic AMP levels, while in our experiments the same drugs were not used as a predose but were given at the time of administration of the inflammatory irritant. Indeed, Zurier et al., (1974) state that “ the time of pre-incubation was critical for reduction of enzyme release to be demonstrated ” in vitro. Another technical point worth noting is that the inhibitory effect of cyclic AMP on enzyme release in vitro can only be demonstrated unequivocally with leucocytes pretreated with the antibiotic cytochalasin B (Zurier et al., 1974). This pretreatment regimen could be strongly criticised following the recent demonstration by Skosey et al., (1974) that cytochalasin B itself inhibits the release of lysosomal enzymes from zymosan-stimulated PMN’s. Bearing in mind these rather rigid and nonphysiologic prerequisites, it could be proposed that the results of such in-vitro experiments might bear no resemblance to the situation occurring in acute inflammation in vivo. Finally, it should be mentioned that whereas our animal experiments involved rats, the in-vitro work of Weissmann and co-workers was
134
D. A. DEPORTER
done with human leucocytes, but whether this species difference is important remains to be determined. The reverse passive Arthus reaction in the rat pleural cavity has already been used to demonstrate that the release of histamine and prostaglandin E, both mediators of prime importance in acute inflammation, but not prostaglandin F, can be significantly reduced by artificially elevating leucocyte cyclic AMP levels (Deporter, Capasso and Willoughby, 1976). These results with histamine supported earlier in-vitro reports in which it was demonstrated that dibutyryl cyclic AMP and/or theophylline significantly decreased the immunologic release of histamine from isolated sensitised rat mast cells following exposure to specific antigen (Kaliner and Austen, 1974; Kimura, Inoue and Honda, 1974). Because cyclic AMP has been shown to inhibit the assembly of isolated microtubular subunits (Goodman et al., 1970) and because intact microtubules are thought to be essential for histamine release from leucocytes (Gillespie and Lichtenstein, 1972), it has been proposed that the inhibitory effect of cyclic AMP on histamine release is mediated by an effect on leucocyte microtubular integrity (see Becker and Henson, 1973). A similar effect on microtubules of PMN’s was proposed by Weissmann’s group to explain their demonstration of reduced lysosomal enzyme release following elevation of PMN cyclic AMP levels in vitro (Zurier et al., 1974). The present in-vivo results and those of our earlier reports (Deporter, et al., 1976; Deporter et al., 1977) raise the following possibilities: (i) Cyclic AMP plays a regulatory role in histamine release from mast cells and/or basophils but not in lysosomal enzyme secretion by PMN’s. This could imply that either microtubules in these different cell types have different sensitivities to cyclic AMP or that microtubules in PMN’s are not involved in lysosomal enzyme discharge in vivo. The latter conclusion tends to be supported by our results with colchicine, an agent known to cause dissolution of microtubules in PMN’s (see Malawista, 1975, for a review). Despite the fact that leucocyte cyclic AMP levels were found to be greatly increased by colchicine, the drug had no effect on extracellular levels of the lysosomal marker, B-glucuronidase. It should be noted, however, that other workers (Hoffstein, Zurier and Weissmann, 1974; Malawista, 1975) have claimed that colchicine reduces lysosomal enzyme release from stimulated human PMN’s in vitro. (ii) Cyclic AMP does not mediate its effects in vivo through disaggregation of microtubules in either inflammatory cell type; this would imply that elevated leucocyte cyclic AMP levels inhibit histamine release by some other unknown mechanism. SUMMARY The pleural cavity of rats was used to study the effect of altering leucocyte cyclic AMP content on the release of B-glucuronidase activity during an immediate hypersensitivity reaction. The effect on intravenous colchicine was also studied. Despite an increase of 135 to 235 per cent. in leucocyte cyclic AMP content
LEUCOCYTE CYCLIC AMP
135
no decrease in B-glucuronidase release was observed. Similarly, colchicine had no effect on enzyme release. It was concluded that the cyclic nucleotides and leucocyte microtubules may have no significant role to play in the release of lysosomal enzymes during acute inflammation in vivo. This investigation was carried out while the author was a post-doctoral research fellow of the Medical Research Council of Canada. The author wishes to thank Professor D. A. Willoughby for his encouragement and advice and the European Biological Research Association for financial support. REFERENCES BECKER,E. L., AND HENSON,P. M. 1973. In vitro studies of immunologically induced secretion of mediators from cells and related phenomena. Adv. Zmmunol., 17, 94 J. D. M., EKINS,R. P., SCHERZI,A. M., AND TAMPION, W. 1971. BROWN,B. L., ALBANO, A simple and sensitive assay method for the measurement of adenosine 3‘,5’-cyclic monophosphate. Biochem. J., 121, 561. COCHRANE, C. G., AND JANOFF, A. 1974. The Arthus reaction: a model of neutrophil and complement-mediated injury. Zn The inflammatory process, edited by B. W. Zweifach, L. Grant and R. T. McCluskey, vol. 111, 2nd ed., Academic Press, New York, p. 85. D. A., CAPASSO, F., AND WILLOUGHBY, D. A. 1976. Effects of modification of DEPORTER, intracellular cyclic AMP levels on the immediate hypersensitivity reaction in vivo. J. Path., 119, 147. DEPORTER, D. A., DIEPPE, P. A., GLATT, M., AND WILLOUGHBY, D. A. 1977. The relation of cyclic AMP levels to phagocytosis and enzyme release in acute inflammation in vivo. J. Path., 121, 129. GILLESPIE, E., AND LICHTENSTEIN, L. M. 1972. Histamine release from human leucocytes: studies with deuterium oxide, colchicine and cytochalasin B. J. Clin. Invest., 51, 2941. D. B., RASMUSSEN, H., DI BELLA,F., AND GUTHROW, C. E. 1970. Cyclic adenoGOODMAN, phosphorylation of isolated microtubule subsine 3’,5’-monophosphate-~timulated units. Proc. Nat. Acad. Sci. U.S.A., 67, 652. HOFFSTEIN, S., ZURIER,R. B., AND WEISSMANN, G. 1974. Mechanisms of lysosomal enzyme release from human leucocytes. 111. Quantitative morphologic evidence for an effect of cyclic nucleotides and colchicine on degranulation. Clin.Zmmuttol. Immunopathol., 3, 201. IGNARRO, L. J. 1974. Regulation of lysosomal enzyme secretion: role of inflammation. Agents and Actions, 4, 241. KALINER,M., AND AUSTEN,K. F. 1974. Cyclic AMP, ATP and reversed anaphylactic histamine release from rat mast cells. J. Zmmunol., 112, 664. KIMURA,Y., INOUE,Y., AND HONDA,H. 1974. Further studies on rat mast cell degranulation by IgE-anti-IgE and the inhibitory effect of drugs related to cyclic AMP. Zmmunology, 26,983. S. E. 1975. The action of colchicine in acute gouty arthritis. Arthr. Rheum., MALAWISTA, 18, 835. S C m T Z , G., HARDMAN, J. G., SCHULTZ,K., BAIRD,C. E., AND SUTHERLAND, E. W. 1973. The importance of calcium ions for the regulation of guanosine 3’,5‘-cyclic monophosphate levels. Proc. Nat. Acad. Sci., 70, 3889. SKOSEY, J. L., DAMGAARD, E., CHOW,D., AND SORENSEN, L. B. 1974. Modification of zymosan-induced release of lysosomal enzymes from human polymorphonuclear leucocytes by cytochalasin B. J. Cell Biol., 62, 625. SMITH, R. J., AND IGNARRO, L. J. 1975. Bioregulation of lysosomal enzyme secretion from human neutrophils: roles of guanosine 3’,5‘-monophosphate and calcium in stimulussecretion coupling. Proc. Nut. Acad. Sci. U.S.A., 72, 108.
136
D. A . DEPORTER
WEJSSMANN, G . , DUKOR, P., AND ZURIER, R. B. 1971. Effect of cyclic AMP on release of lysosomal enzymes from phagocytes. Nature, New Biology, 231, 131. WEISSMANN, G., ZURIER, R. B., SPIELER, P. J., AND GOLDSTEIN, I. M. 1971. Mechanisms of lysosomal enzyme release from leucocytes exposed to immune complexes and other particles. J. Exper. Med., 134,149s. YAMAMOTO, S., DUNN,C. J., DEPORTER, D. A., CAPASSO, F., WILLOUGHBY, D. A., AND HUSKISSON, E. C. 1975. A model for the quantitative study of Arthus (immunologic) hypersensitivity in rats. Agents and Actions, 5 , 374. ZURIER,R. B., WEISSMANN, G., HOFFSTEIN, S., KAMMERMAN, S., AND TAI, H. H. 1974. Mechanisms of lysosomal enzyme release from human leucocytes. 11. Effects of C-AMP and C-GMP, autonomic agonists and agents which affect microtubule function. J. Clin. Invest., 53, 297.
Journal of Pathology Vol. 123 No. 3 C Y C L I C A M P A N D T H E M E C H A N I S M O F LEUCOCYTE LYSOSOMAL ENZYME RELEASE D U R I N G A N IMMEDIATE HYPERSENSITIVITY REACTION IN VIVO
D. A. DEPORTER* Department of Experimental Pathology, St Bartholomew’s Hos&nl Medical School, London, England
I T is generally recognised that an insight into the control mechanisms involved in the release of lysosomal enzymes from inflammatory leucocytes could be of considerable benefit in the treatment of inflammatory diseases such as crystalinduced arthopathies and rheumatoid arthritis. Recent studies from several laboratories have implicated the cyclic nucleotides, adenosine 3’,5’-cyclic monophosphate (cyclic AMP) and guanosine 3’, 5’-cyclic monophosphate (cyclic GMP), in the control of lysosomal enzyme release from polymorphonuclear leucocytes (PMN’s). It has been demonstrated that the discharge of these potentially destructive acid hydrolases from isolated PMN’s in response to a variety of stimuli, including Ag-Ab complexes can be inhibited by artificially elevating PMN cyclic AMP levels or enhanced by increasing their cyclic GMP levels (Weissmann et al., 1971; Ignarro, 1974; Zurier et al., 1974). However, these results have not as yet been supported by suitable animal experiments. In an earlier study we presented evidence favouring a lack of correlation between lysosomal enzyme release and leucocyte cyclic AMP levels during crystal-induced (Ca pyrophosphate) pleurisy in rats (Deporter et al., 1976). We have now repeated these experiments using a different model of acute inflammation, namely a reverse passive Arthus reaction in the pleural cavity of rats (Yamamoto et al., 1975) and the results comprise the present report. MATERIALS AND
METHODS
Reverse passive Arthus reactions were produced in the pleural cavities of male Wistar rats (Yamamoto et al., 1975) and modified with theophylline and/or dibutyryl cyclic AMP administered as previously described with pyrophosphate-induced pleurisy (Deporter et al., 1976). Received 10 Dec. 1976; accepted 17 Jan. 1977. Division of Biological Sciences, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, MSG lG6, Canada.
* Present address: I. PATH.-VOL.
123 (1977)
129
I
D . B. DEPORTER
130
Cyclic AMP estimations were performed using the method of Brown ef al., (1971). Assays of B-glucuronidase and lactate dehydrogenase (LDH) activities were as before (Deporter ef a!., 1976). In some experiments the effect of elevating leucocyte AMP levels with theophylline and dibutyryl cyclic AMP on leucocyte cyclic GMP levels was studied. Leucocytes from control and drug-treated pleural effusions were fixed in 1 per cent. perchloric acid, sonicated for 30 s and centrifuged at 10,OOOg for 15 min. The clear supernatants were adjusted to pH 7.0 with KOH and recentrifuged. The clear supernatants from this second centrifugation were each ~ acid. Cyclic applied to a separate 1 ml column of Biorad equilibrated with 0 . 1 formic AMP was eluted with 2N formic acid while cyclic GMP was eluted with 4N formic acid.
Control
dC-amp
Both
Colchicine
FIG. 1.-Effect
of intrapleurally administered dibutyryl cyclic AMP (5 mM), intrapleurally administered dibutyryl cyclic AMP with theophylline (5 mM each) and intravenously administered colchicine (0.2 mg/kg) on leucocyte cyclic AMP concentration at 3 hr following the onset of an intrapleural reverse passive Arthus reaction. Values are the mean of 12 experiments.
Following freeze-drying of the eluates, the residues were assayed for cyclic G M P using a commercially available assay kit (Collaborative Research Inc., Waltham, Mass.) and for cyclic AMP as described above. In other animals the effects of colchicine on leucocyte cyclic AMP levels and lysosomal enzyme discharge during the reverse passive Arthus reaction were assessed. Colchicine (BDH, Poole, Dorset) was given intravenously (0.2 mg per kg, in saline 1 hr before eliciting the intrapleural reaction.
RESULTS Effect of dibutyryl cyclic AMP with and without theophylline on leucocyte cyclic AMP levels Administration of 5 mM dibutyryl cyclic AMP intrapleurally at the time of onset of the intrapleural Arthus reaction produced a 135 per cent. increase
LEUCOCYTE CYCLIC AMP
131
in leucocyte cyclic AMP concentration as compared with leucocytes from control reactions. When 5 mM dibutyryl cyclic AMP and 5 mM theophylline were given together, a 235 per cent. increase in leucocyte cyclic AMP content was observed (fig. 1). 25
20
1
10
5
dC-amp
Control
Both
FIG. 2.-Effect of dibutyryl cyclic AMP alone and in combination with theophylline (both) on the percentage B-glucuronidase activity released in the Arthus-induced pleural effusions. Values are the mean of 12 experiments. extracellular enzyme activity x 100 percentage released = cellular extracellular activity ~
+
TABLEI Efect of dibutyryl cyclic AMP with or without theophylline on extracellular L D H levels; the values represent the mem of twelve experiments Control
Percentage LDH released 14.5f2.0
dibutyryl cyclic AMP
20.0f2.9
dibutyryl cyclic AMP theophylline
183+4.9
+
Effect of dibutyryl cyclic AMP with and without theophylline on the percentage leucocyte lysosomal B-glucuronidase and leucocyte lactate dehydrogenase activities released. Administration of dibutyryl cyclic AMP produced no reduction in the amount of B-glucuronidase released into the 3-hr Arthus-induced pleural effusions. When dibutyryl cyclic AMP and theophylline were both injected intrapleurally at the time of onset of the Arthus reaction, there was a 30 per cent. increase in enzyme release (fig. 2). The LDH results were slightly different (table 1). The smallest amounts of LDH were released in control reactions
D. A . DEPORTER
132
but in all three groups studied the percentage LDH released was never more than 25 per cent. of the total leucocyte LDH content.
EfSect of administering dibutyryl cyclic AMP and theophylline on leucocyte cyclic GMP levels The results of these experiments are shown in table 11. Cyclic AMP and cyclic GMP levels were assayed for the same pleural exudates after separation TABLEI1 EfSect of 5 mM dibutyryl cyclic AMP and 5 mM theophylline on leucocyte cyclic GMP and cyclic A M P levels from the same pleural reactions; the values represent the mean of twelve experiments Cyclic GMP/lOg cells Cyclic AMP/lOs cells Control Arthus reaction
0.83 pmoles
65 pmoles
Drug-modified Arthus reaction
1.3 pmoles
247 pmoles
of the two nucleotides on mini-columns of Biorad. Whereas the treatment produced a 280 per cent. increase in leucocyte cyclic AMP concentration it produced a 57 per cent. increase in leucocyte cyclic GMP content. 200
20
1150
~
2
EL
.-
5
0%
I
I
W
tT rn
15 1100
-I
E C
g
10
Y
%
'.SO 5
FIG. 3.--Effect of colchicine on the extracellular concentration of B-glucuronidase activity (left side) and of the total extracellular B-glucuronidase activity (right side). Values are the mean of 12 experiments. Cross-hatch=colchicine; diagonal stripe=control.
Effect of colchicine on B-glucuronidase released into pleural effusions As can be seen in fig. 3, pretreatment of animals with intravenous colchicine produced no decrease in the amount of B-glucuronidase released into the Arthus-
LEUCOCYTE CYCLIC AMP
133
induced pleural effusions. In contrast to this lack of effect of colchicine on enzyme release, the same pretreatment produced a 154 per cent. increase in leucocyte cyclic AMP concentration (fig. 1). DISCUSSION
We have previously demonstrated an apparent lack of inhibition of lysosomal enzyme discharge from leucocytes following therapeutic elevation of cellular cyclic AMP levels during pyrophosphate pleurisy in rats (Deporter et al., 1977). The present results in a different model of acute inflammation, an immediate hypersensitivity reaction in the pleural cavity of rats, provide further evidence for this conclusion. Further, they tend to dispel any argument suggesting that the earlier results were due primarily to an effect of free calcium ions. It might have been argued, for example, following the work of Smith and Ignarro (1975) and others (Schultz et al., 1973), that whereas the drugs used (dibutyryl cyclic AMP and theophylline) produced a marked increase in leucocyte cyclic AMP levels, the extra free calcium might have produced a much larger increase in cyclic GMP with concomitant lysosomal enzyme discharge. Administration of dibutyryl cyclic AMP with or without theophylline into the pleural cavity of rats at the time of initiation of an immediate hypersensitivity reaction in the same site produced a 135 to 235 per cent. increase in leucocyte cyclic AMP levels but had no significant effect on secretion of the lysosomal hydrolase, B-glucuronidase. This observation that cyclic AMP does not monitor lysosomal release in vivo is in direct opposition to the in-vitro work of Weissmann’s group (e.g., Weissmann, Dukor and Zurier, 1971). The reason for this lack of agreement is not readily apparent. These other workers did use concentrations of dibutyryl cyclic AMP and theophylline approximating our own dose regimens. Furthermore, for the most part they utilised mixed populations of leucocytes rather than purified PMN’s which again corresponds well to the population of cells present in a pleural effusion. One important point of diflerence, however, is that in the in-vitro experiments the leucocytes were pre-incubated with agents designed to increase cyclic AMP levels, while in our experiments the same drugs were not used as a predose but were given at the time of administration of the inflammatory irritant. Indeed, Zurier et al., (1974) state that “ the time of pre-incubation was critical for reduction of enzyme release to be demonstrated ” in vitro. Another technical point worth noting is that the inhibitory effect of cyclic AMP on enzyme release in vitro can only be demonstrated unequivocally with leucocytes pretreated with the antibiotic cytochalasin B (Zurier et al., 1974). This pretreatment regimen could be strongly criticised following the recent demonstration by Skosey et al., (1974) that cytochalasin B itself inhibits the release of lysosomal enzymes from zymosan-stimulated PMN’s. Bearing in mind these rather rigid and nonphysiologic prerequisites, it could be proposed that the results of such in-vitro experiments might bear no resemblance to the situation occurring in acute inflammation in vivo. Finally, it should be mentioned that whereas our animal experiments involved rats, the in-vitro work of Weissmann and co-workers was
134
D. A. DEPORTER
done with human leucocytes, but whether this species difference is important remains to be determined. The reverse passive Arthus reaction in the rat pleural cavity has already been used to demonstrate that the release of histamine and prostaglandin E, both mediators of prime importance in acute inflammation, but not prostaglandin F, can be significantly reduced by artificially elevating leucocyte cyclic AMP levels (Deporter, Capasso and Willoughby, 1976). These results with histamine supported earlier in-vitro reports in which it was demonstrated that dibutyryl cyclic AMP and/or theophylline significantly decreased the immunologic release of histamine from isolated sensitised rat mast cells following exposure to specific antigen (Kaliner and Austen, 1974; Kimura, Inoue and Honda, 1974). Because cyclic AMP has been shown to inhibit the assembly of isolated microtubular subunits (Goodman et al., 1970) and because intact microtubules are thought to be essential for histamine release from leucocytes (Gillespie and Lichtenstein, 1972), it has been proposed that the inhibitory effect of cyclic AMP on histamine release is mediated by an effect on leucocyte microtubular integrity (see Becker and Henson, 1973). A similar effect on microtubules of PMN’s was proposed by Weissmann’s group to explain their demonstration of reduced lysosomal enzyme release following elevation of PMN cyclic AMP levels in vitro (Zurier et al., 1974). The present in-vivo results and those of our earlier reports (Deporter, et al., 1976; Deporter et al., 1977) raise the following possibilities: (i) Cyclic AMP plays a regulatory role in histamine release from mast cells and/or basophils but not in lysosomal enzyme secretion by PMN’s. This could imply that either microtubules in these different cell types have different sensitivities to cyclic AMP or that microtubules in PMN’s are not involved in lysosomal enzyme discharge in vivo. The latter conclusion tends to be supported by our results with colchicine, an agent known to cause dissolution of microtubules in PMN’s (see Malawista, 1975, for a review). Despite the fact that leucocyte cyclic AMP levels were found to be greatly increased by colchicine, the drug had no effect on extracellular levels of the lysosomal marker, B-glucuronidase. It should be noted, however, that other workers (Hoffstein, Zurier and Weissmann, 1974; Malawista, 1975) have claimed that colchicine reduces lysosomal enzyme release from stimulated human PMN’s in vitro. (ii) Cyclic AMP does not mediate its effects in vivo through disaggregation of microtubules in either inflammatory cell type; this would imply that elevated leucocyte cyclic AMP levels inhibit histamine release by some other unknown mechanism. SUMMARY The pleural cavity of rats was used to study the effect of altering leucocyte cyclic AMP content on the release of B-glucuronidase activity during an immediate hypersensitivity reaction. The effect on intravenous colchicine was also studied. Despite an increase of 135 to 235 per cent. in leucocyte cyclic AMP content
LEUCOCYTE CYCLIC AMP
135
no decrease in B-glucuronidase release was observed. Similarly, colchicine had no effect on enzyme release. It was concluded that the cyclic nucleotides and leucocyte microtubules may have no significant role to play in the release of lysosomal enzymes during acute inflammation in vivo. This investigation was carried out while the author was a post-doctoral research fellow of the Medical Research Council of Canada. The author wishes to thank Professor D. A. Willoughby for his encouragement and advice and the European Biological Research Association for financial support. REFERENCES BECKER,E. L., AND HENSON,P. M. 1973. In vitro studies of immunologically induced secretion of mediators from cells and related phenomena. Adv. Zmmunol., 17, 94 J. D. M., EKINS,R. P., SCHERZI,A. M., AND TAMPION, W. 1971. BROWN,B. L., ALBANO, A simple and sensitive assay method for the measurement of adenosine 3‘,5’-cyclic monophosphate. Biochem. J., 121, 561. COCHRANE, C. G., AND JANOFF, A. 1974. The Arthus reaction: a model of neutrophil and complement-mediated injury. Zn The inflammatory process, edited by B. W. Zweifach, L. Grant and R. T. McCluskey, vol. 111, 2nd ed., Academic Press, New York, p. 85. D. A., CAPASSO, F., AND WILLOUGHBY, D. A. 1976. Effects of modification of DEPORTER, intracellular cyclic AMP levels on the immediate hypersensitivity reaction in vivo. J. Path., 119, 147. DEPORTER, D. A., DIEPPE, P. A., GLATT, M., AND WILLOUGHBY, D. A. 1977. The relation of cyclic AMP levels to phagocytosis and enzyme release in acute inflammation in vivo. J. Path., 121, 129. GILLESPIE, E., AND LICHTENSTEIN, L. M. 1972. Histamine release from human leucocytes: studies with deuterium oxide, colchicine and cytochalasin B. J. Clin. Invest., 51, 2941. D. B., RASMUSSEN, H., DI BELLA,F., AND GUTHROW, C. E. 1970. Cyclic adenoGOODMAN, phosphorylation of isolated microtubule subsine 3’,5’-monophosphate-~timulated units. Proc. Nat. Acad. Sci. U.S.A., 67, 652. HOFFSTEIN, S., ZURIER,R. B., AND WEISSMANN, G. 1974. Mechanisms of lysosomal enzyme release from human leucocytes. 111. Quantitative morphologic evidence for an effect of cyclic nucleotides and colchicine on degranulation. Clin.Zmmuttol. Immunopathol., 3, 201. IGNARRO, L. J. 1974. Regulation of lysosomal enzyme secretion: role of inflammation. Agents and Actions, 4, 241. KALINER,M., AND AUSTEN,K. F. 1974. Cyclic AMP, ATP and reversed anaphylactic histamine release from rat mast cells. J. Zmmunol., 112, 664. KIMURA,Y., INOUE,Y., AND HONDA,H. 1974. Further studies on rat mast cell degranulation by IgE-anti-IgE and the inhibitory effect of drugs related to cyclic AMP. Zmmunology, 26,983. S. E. 1975. The action of colchicine in acute gouty arthritis. Arthr. Rheum., MALAWISTA, 18, 835. S C m T Z , G., HARDMAN, J. G., SCHULTZ,K., BAIRD,C. E., AND SUTHERLAND, E. W. 1973. The importance of calcium ions for the regulation of guanosine 3’,5‘-cyclic monophosphate levels. Proc. Nat. Acad. Sci., 70, 3889. SKOSEY, J. L., DAMGAARD, E., CHOW,D., AND SORENSEN, L. B. 1974. Modification of zymosan-induced release of lysosomal enzymes from human polymorphonuclear leucocytes by cytochalasin B. J. Cell Biol., 62, 625. SMITH, R. J., AND IGNARRO, L. J. 1975. Bioregulation of lysosomal enzyme secretion from human neutrophils: roles of guanosine 3’,5‘-monophosphate and calcium in stimulussecretion coupling. Proc. Nut. Acad. Sci. U.S.A., 72, 108.
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WEJSSMANN, G . , DUKOR, P., AND ZURIER, R. B. 1971. Effect of cyclic AMP on release of lysosomal enzymes from phagocytes. Nature, New Biology, 231, 131. WEISSMANN, G., ZURIER, R. B., SPIELER, P. J., AND GOLDSTEIN, I. M. 1971. Mechanisms of lysosomal enzyme release from leucocytes exposed to immune complexes and other particles. J. Exper. Med., 134,149s. YAMAMOTO, S., DUNN,C. J., DEPORTER, D. A., CAPASSO, F., WILLOUGHBY, D. A., AND HUSKISSON, E. C. 1975. A model for the quantitative study of Arthus (immunologic) hypersensitivity in rats. Agents and Actions, 5 , 374. ZURIER,R. B., WEISSMANN, G., HOFFSTEIN, S., KAMMERMAN, S., AND TAI, H. H. 1974. Mechanisms of lysosomal enzyme release from human leucocytes. 11. Effects of C-AMP and C-GMP, autonomic agonists and agents which affect microtubule function. J. Clin. Invest., 53, 297.
