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Some Aspects of Anti-Inflammatory Action of Immunosuppressive Drugs
SOME
ASPECTS OF
Wataru
OF
ANTI-INFLAMMATORY
IMMUNOSUPPRESSIVE
TSUKADA,
Takeshi
AKIMOTO
ACTION DRUGS
and Yutaka
MIZUSHIMA*
Laboratory of Pharmacology, Research Institute, Daiiclri Seiyaku Co., Ltd., Edogawa-ku, Tokyo and Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo,* Tokyo, Japan Accepted March 6, 1974
Abstract--Two phase rat paw inflammation was induced by injection of carrageenan into the paw of rats inoculated with adjuvant (adjuvant-carrageenan-induced inflam mation, ACII). The model was utilized to analyze the anti-inflammatory action of immunosuppressive drugs. Immunosuppressive drugs intensively inhibited the prolonged phase of ACII, which is considered to occur through the same mecha nism as that of adjuvant arthritis. The inhibition was not attributed to immuno suppressive effects nor simple anti-inflammatory effects of the drugs. Immunosup pressive drugs appear to suppress certain biologic activities of lymphoid cells which act as a trigger to induce the prolonged phase.
Two phase rat paw inflammation (acute and prolonged) was induced by the injec tion of carrageenan into the paw of rats inoculated with adjuvant 3 or more days previ ously (adjuvant-carrageenan-induce(I inflammation, ACII). The prolonged phase of AC11 is considered to occur through a mechanism common to that of adjuvant arthritis as discussed in the previous study (1). Since beneficial effects of immunosuppressive drugs on adjuvant arthritis, rheumatoid arthritis and other connective tissue diseases have been frequently reported, the effects of immunosuppressive drugs on ACII were examined in an attempt to clarify the mode of action of the drugs. MATERIALSAND METHODS Random-bred female rats of the Sprague-Dawleystrain aged from 8 to I I weeks and weighing 200 to 280 g were used. To produce carrageenan paw edema, 0.1 nil of 1 carrageenan suspension in saline was injected subplantarly into the left hindpaw. Car rageenan was supplied by Nitto Kaiso, Tokyo. ACII was produced as follows. A 0.1 ml liquid paraffin (Merck, Germany) con taining 0.6 mg of heat-killed Mycobacterium butyricum (Difco) was injected i.d. into the basal part of the tail. A 0.1 nil of I 1.5 carrageenan suspension was injected s.c. into the left hindpaw of the rats 6 or 9 days after the adjuvant inoculation. Concentration of carrageenan was determined according to the swelling index 3 hr after the carrageenan injection into the controls reached above 80 on an average. The volume of the left hind paw was measured after the method described by Van Arman et al. (2). The swelling index of the inflamed paw was calculated from the following formula.
S Vol. of swollen paw Vol. of paw before injection welling Index Vol. of paw before injection x 100 Percentage inhibition by drugs was then calculated from the swelling index. Difference in the swelling index between ACII and carrageenan edema in non-treated animals was read as 100",, for calculating the drug effects on the prolonged phase of ACII. The drugs used and the method of administration were as follows. Cortisol (hy drocortisone acetate, Merck), 20 mg/kg, s.c. Methotrexate (Lederle), 0.3 mg/kg, i.p. Cyclophosphamide (Shionogi, Osaka), 10 mg/kg, i.p. 6-Mercaptopurine (6MP, Takeda, Osaka), 20 mg/kg, i.p. Azathioprine (Wellcome), 30 mg/kg, i.m. Cortisol, methot rexate and cyclophosphamide were dissolved or suspended in saline. Azathioprine and 6MP were dissolved with NaOH and diluted with saline. A dose of 0.5 ml/ 100g body weight of the drug solution was injected. The control animals were given the same vol ume of saline. The dorsal portion of the inflamed paw was excised for histological studies. The tissues were fixed in 10'/ formalin and embedded in paraffin. Sections were stained with haematoxylin and eosin. RESULTS E ei cts of immunosuppressirrc
drugs and cortisol
on carrageenan
pan' edema in rats
Methotrexate and cortisol were given 3 times, 24 hr and I hr before and 24 hr after the carrageenan injection.
As shown in Fig. I methotrexate did not influence carrage
enan edema, while cortisol suppressed the swelling.
Fn;,
1.
Effects
of
methotrexate
and
cortisol
on
Neither cyclophosphamide
carrageenan
paw
edema
in
rats.
nor
TABLE 1. Effects
of immunosuppressive
drugs
on
carrageenan
paw
edema
in rats.
Drugs were administered 24 hr and 1 hr before carrageenan injection, and effects were measured 3 hr after. * Significance (P<0 .05) in relation to control group.
azathioprine theless
suppressed
significantly
Suppression
carrageenan
(Table
and cortisol
which did not influence
prolonged
phase.
given
any
Cortisol
effects day
are
experimental
on
6MP
inhibited
it slightly
but never
the acute
suppressed
ACII
of
summarized day
drugs
were given to the rats 3 times as shown
thotrexate
Inhibitory
while
1).
of ACII by intnuunosuppressirc
Methotrexate
experimental
edema,
did
methotrexatc in
not
phase of ACII completely
both the acute
Table
suppress
and
and prolonged
cyclophosphamide
2.
Methotrexate
the
early
phase
and of
ACII.
in Fig. 2.
Me
suppressed phase
the
of ACII.
given
on
each
cyclophosphamide Methotrexate
TABLE2. Effects of immunosuppressive inflammation (ACID in rats.
drugs on adjuvant-carrageenan-induced
ACII was induced by the injection of carrageenan on 6th experimental day into th( paw of rats inoculated with adjuvant on 0 day. Drug effects on the acute and pro longed phase of ACII were measured 3 hr and 72 hr after the carrageenan injection respectively. a) CP : Cyclophosphamide, 10 mg, kg. MTX : Methotrexate, 0.3 mg;'kg 6MP : 20 nag kg. AZP : Azathioprine, 30 mg' kg. *Significance (P--;0.05) in relation to control group.
T.ABLe3. Effects of methotrexate 1n rQ'ka) nn ACIL
(MTX, 0.3 nag kg) and cyclophospharnide
(CP,
ACIT was induced by the injection of carrageenan on 9th experimental day into the paw of rats inoculated with adjuvant on 0 day. Drug effects on acute and pro longed phase of AC[I were measured 3 hr and 72 hr after the carrageenan injection, respectively. * Significance (P "0 .05) in relation to control group.
and cyclophosphamide given shortly before the carrageenan injection, i.e. about the 5th experimental day considerably suppressed the prolonged phase. Drugs given on other days did not significantly inhibit the prolonged phase. 6-Mercaptopurine and azathio prine inhibited the prolonged phase of ACII. The inhibitory effects, however, appeared to be weak at least in the dose employed as compared with effects of cyclophosphamide and methotrexate. When carrageenan was injected 9 days after the adjuvant inoculation, similar find ings were obtained (Table 3). Methotrexate and cyclophosphamide given around the carrageenan injection were also effective on the prolonged phase. As shown in Table 2, the acute phase of ACII was slightly suppressed by 6MP. Effect of methotrexate on cell infiltration Five rats were injected with carrageenan alone and histological studies were carried out. The dorsal potrion of the inflamed paws consisting of the skin and subcutaneous connective tissues was excised 4, 24 or 48 hr after the carrageenan injection. Cellular infiltration in the inflamed subcutaneous connective tissues was evident in all preparations. The dominant cells in the inflamed lesions were polymorphonuclear cells at 4 hr and mo nonuclear cells, mainly macrophages and lymphocytes at 24 and 48 hr. Five rats were treated with methotrexate in a dose of 0.3 mg,`kg 24 hr and 1 hr before the carrageenan injection. The degree of cellular infiltration and type of dominant cells was however unchanged. DISCUSSION The role of cellular hypersensitivity in the pathogenesis of adjuvant arthritis has been well demonstrated. Our observations herein suggest that the sensitized lymphoid cells which migrate into the site of the carrageenan-induced inflammation in rats inocu lated with adjuvant act as a trigger to produce the prolonged phase of ACII. The prolonged phase began to occur 1-2 days after the carrageenan injection, which was corresponds roughly to the time of lymphoid cell migration into the inflamed paw. Many immunosuppressive drugs suppress primary immune response and experi mentally induced delayed hypersensitivity in humans as well as in animals. However several investigators have pointed out that treatment with immunosuppressive drugs often improved clinical features without alterating established immune response. Impro vement of symptoms by immunosuppressive drugs in rheumatoid arthritis and systemic lupus erythematosus does not appear to be related to the changes in titer of immunologic reaction or amount of immunoglobulin (3, 4, 5). Thus, immunosuppressive drugs ap pear to have a certain type of anti-inflammatory action in addition to their immunosup pressive effects. 6-Mercaptopurine was found to alter the inflammatory cycle in rabbits , virtually eliminating the participation of mononuclear cells (6). Hersh et al . (7) studied the ef fects of nme!hotre-yate and 6MP on the local inhainnatory response in humans and also observed an inhibition of the mononuclear cell phase. It has also been reported that
the anti-inflammatory effects of 6MP are due to suppression of a bone marrow response to local inflammation (8). In the present study, the prolonged phase of ACII was markedly inhibited by im munosuppressive drugs given shortly before the carrageenan injection. For the follow ing reasons it is unlikely that the inhibition was due to immunosuppressiveeffects of the drugs: 1) The drugs given before 5th (Table 2) or 6th (Table 3) post-inoculation day did not suppress the prolonged inflammation, rather the immunosuppressiveaction was usually more prominent when the drugs had been given shortly after or just before antigen stimulation (9). 2) As reported previously, the prolonged phase was produced on the 5th post-inoculation day when carrageenan was injected on 3rd day (1). In this study, however, immunosuppressive drugs given from 5th to 7th or 8th to 10th post-inocula tion days intensively suppressed the prolonged phase. Since acute inflammation such as thermal injury and turpentine-induced pleurisy was suppressed by 6MP, methotrexate and cyclophosphamide (10), the simple anti-in flammatory action of immunosuppressive drugs was suggested. However carrageenan edema and the early phase of ACII were not suppressed by these drugs in this study. Therefore, the suppression of the prolonged phase of ACII by immunosuppressivedrugs was not considered to be due to the non-specific anti-inflammatory action of the drugs. Moreover methotrexate did not inhibit cellular migration into the inflamed lesion. As discussed above, the mechanism of inhibitory effects of immunosuppressivedrugs on the prolonged phase of ACII remains unclear, however, since lymphoid cells are con sidered to mediate the prolonged phase of ACII and immunosuppressive drugs affect some lymphoid cells, the suppression of the prolonged phase by the drugs appears at tributable to the action of drugs on certain biologic activities of sensitized and,'or non sensitized lymphoid cells which migrate into the rats paw. The effects of these drugs on the release of lymphocytic mediators or rnacrophage-lymphocyte interaction have yet to be determined. It is thus concluded that certain activities of immunosuppressive drugs other than immunosuppressive and simple anti-inflammatory actions play a role in the mechanism of action of these drugs. Acknoltwleci±,enients: Thanks are due to Mr. M. Tsubokawa, Mr. A. Yamada and Mr. M. Kato for skillful technical assistance. REFERENCES 1) MIZUSHIMA, Y., TSUKADA, W. ANDAKIMOTO, T.: Ann. rheum.Dis. 29, 178(1970) 2) VANARMAN, C.G., BEGANY, A.J., MILLER, L.M. ANDPLESS, H.H.: J. Pharmacol.exp. Ther. 150, 328 (1965) 3) ALEPA, F.P., ZVAIFLER, N.J. ANDSLINWINSKI, A.J.: Arthr. and Rheum.13, 754 (1970) 4) SWANSON, M.A., ANDSCHWARTZ, R.S.: New. Engl. J. Med. 277, 163 (1967) 5) DADSON, W.H. ANDBENNETT, J.C.: J. clin. Pharmacol.9, 251 (1969) 6) PAGE,A.R., CONDIE, R.M. ANDGOOD,R.A.: Am. J. Path. 40, 519 (1962) 7) HERSH, E.M., WONG,V.G. ANDFREIREICH, E.J.: Blood,27, 38 (1966) 8) HURD,E.R. ANDZIFF,R.: J. exp. Med.128, 785 (1968) 9) MAKINODAN, T., SANTOS, G.W. ANDQUINN,R.P.: Pharmacol.Rev. 22, 189 (1970) 10) STEVENS, J.E. ANDWILLOUGHBY, D.A.: J. Path. 97, 367 (1969)
ASPECTS OF
Wataru
OF
ANTI-INFLAMMATORY
IMMUNOSUPPRESSIVE
TSUKADA,
Takeshi
AKIMOTO
ACTION DRUGS
and Yutaka
MIZUSHIMA*
Laboratory of Pharmacology, Research Institute, Daiiclri Seiyaku Co., Ltd., Edogawa-ku, Tokyo and Department of Medicine and Physical Therapy, Faculty of Medicine, University of Tokyo,* Tokyo, Japan Accepted March 6, 1974
Abstract--Two phase rat paw inflammation was induced by injection of carrageenan into the paw of rats inoculated with adjuvant (adjuvant-carrageenan-induced inflam mation, ACII). The model was utilized to analyze the anti-inflammatory action of immunosuppressive drugs. Immunosuppressive drugs intensively inhibited the prolonged phase of ACII, which is considered to occur through the same mecha nism as that of adjuvant arthritis. The inhibition was not attributed to immuno suppressive effects nor simple anti-inflammatory effects of the drugs. Immunosup pressive drugs appear to suppress certain biologic activities of lymphoid cells which act as a trigger to induce the prolonged phase.
Two phase rat paw inflammation (acute and prolonged) was induced by the injec tion of carrageenan into the paw of rats inoculated with adjuvant 3 or more days previ ously (adjuvant-carrageenan-induce(I inflammation, ACII). The prolonged phase of AC11 is considered to occur through a mechanism common to that of adjuvant arthritis as discussed in the previous study (1). Since beneficial effects of immunosuppressive drugs on adjuvant arthritis, rheumatoid arthritis and other connective tissue diseases have been frequently reported, the effects of immunosuppressive drugs on ACII were examined in an attempt to clarify the mode of action of the drugs. MATERIALSAND METHODS Random-bred female rats of the Sprague-Dawleystrain aged from 8 to I I weeks and weighing 200 to 280 g were used. To produce carrageenan paw edema, 0.1 nil of 1 carrageenan suspension in saline was injected subplantarly into the left hindpaw. Car rageenan was supplied by Nitto Kaiso, Tokyo. ACII was produced as follows. A 0.1 ml liquid paraffin (Merck, Germany) con taining 0.6 mg of heat-killed Mycobacterium butyricum (Difco) was injected i.d. into the basal part of the tail. A 0.1 nil of I 1.5 carrageenan suspension was injected s.c. into the left hindpaw of the rats 6 or 9 days after the adjuvant inoculation. Concentration of carrageenan was determined according to the swelling index 3 hr after the carrageenan injection into the controls reached above 80 on an average. The volume of the left hind paw was measured after the method described by Van Arman et al. (2). The swelling index of the inflamed paw was calculated from the following formula.
S Vol. of swollen paw Vol. of paw before injection welling Index Vol. of paw before injection x 100 Percentage inhibition by drugs was then calculated from the swelling index. Difference in the swelling index between ACII and carrageenan edema in non-treated animals was read as 100",, for calculating the drug effects on the prolonged phase of ACII. The drugs used and the method of administration were as follows. Cortisol (hy drocortisone acetate, Merck), 20 mg/kg, s.c. Methotrexate (Lederle), 0.3 mg/kg, i.p. Cyclophosphamide (Shionogi, Osaka), 10 mg/kg, i.p. 6-Mercaptopurine (6MP, Takeda, Osaka), 20 mg/kg, i.p. Azathioprine (Wellcome), 30 mg/kg, i.m. Cortisol, methot rexate and cyclophosphamide were dissolved or suspended in saline. Azathioprine and 6MP were dissolved with NaOH and diluted with saline. A dose of 0.5 ml/ 100g body weight of the drug solution was injected. The control animals were given the same vol ume of saline. The dorsal portion of the inflamed paw was excised for histological studies. The tissues were fixed in 10'/ formalin and embedded in paraffin. Sections were stained with haematoxylin and eosin. RESULTS E ei cts of immunosuppressirrc
drugs and cortisol
on carrageenan
pan' edema in rats
Methotrexate and cortisol were given 3 times, 24 hr and I hr before and 24 hr after the carrageenan injection.
As shown in Fig. I methotrexate did not influence carrage
enan edema, while cortisol suppressed the swelling.
Fn;,
1.
Effects
of
methotrexate
and
cortisol
on
Neither cyclophosphamide
carrageenan
paw
edema
in
rats.
nor
TABLE 1. Effects
of immunosuppressive
drugs
on
carrageenan
paw
edema
in rats.
Drugs were administered 24 hr and 1 hr before carrageenan injection, and effects were measured 3 hr after. * Significance (P<0 .05) in relation to control group.
azathioprine theless
suppressed
significantly
Suppression
carrageenan
(Table
and cortisol
which did not influence
prolonged
phase.
given
any
Cortisol
effects day
are
experimental
on
6MP
inhibited
it slightly
but never
the acute
suppressed
ACII
of
summarized day
drugs
were given to the rats 3 times as shown
thotrexate
Inhibitory
while
1).
of ACII by intnuunosuppressirc
Methotrexate
experimental
edema,
did
methotrexatc in
not
phase of ACII completely
both the acute
Table
suppress
and
and prolonged
cyclophosphamide
2.
Methotrexate
the
early
phase
and of
ACII.
in Fig. 2.
Me
suppressed phase
the
of ACII.
given
on
each
cyclophosphamide Methotrexate
TABLE2. Effects of immunosuppressive inflammation (ACID in rats.
drugs on adjuvant-carrageenan-induced
ACII was induced by the injection of carrageenan on 6th experimental day into th( paw of rats inoculated with adjuvant on 0 day. Drug effects on the acute and pro longed phase of ACII were measured 3 hr and 72 hr after the carrageenan injection respectively. a) CP : Cyclophosphamide, 10 mg, kg. MTX : Methotrexate, 0.3 mg;'kg 6MP : 20 nag kg. AZP : Azathioprine, 30 mg' kg. *Significance (P--;0.05) in relation to control group.
T.ABLe3. Effects of methotrexate 1n rQ'ka) nn ACIL
(MTX, 0.3 nag kg) and cyclophospharnide
(CP,
ACIT was induced by the injection of carrageenan on 9th experimental day into the paw of rats inoculated with adjuvant on 0 day. Drug effects on acute and pro longed phase of AC[I were measured 3 hr and 72 hr after the carrageenan injection, respectively. * Significance (P "0 .05) in relation to control group.
and cyclophosphamide given shortly before the carrageenan injection, i.e. about the 5th experimental day considerably suppressed the prolonged phase. Drugs given on other days did not significantly inhibit the prolonged phase. 6-Mercaptopurine and azathio prine inhibited the prolonged phase of ACII. The inhibitory effects, however, appeared to be weak at least in the dose employed as compared with effects of cyclophosphamide and methotrexate. When carrageenan was injected 9 days after the adjuvant inoculation, similar find ings were obtained (Table 3). Methotrexate and cyclophosphamide given around the carrageenan injection were also effective on the prolonged phase. As shown in Table 2, the acute phase of ACII was slightly suppressed by 6MP. Effect of methotrexate on cell infiltration Five rats were injected with carrageenan alone and histological studies were carried out. The dorsal potrion of the inflamed paws consisting of the skin and subcutaneous connective tissues was excised 4, 24 or 48 hr after the carrageenan injection. Cellular infiltration in the inflamed subcutaneous connective tissues was evident in all preparations. The dominant cells in the inflamed lesions were polymorphonuclear cells at 4 hr and mo nonuclear cells, mainly macrophages and lymphocytes at 24 and 48 hr. Five rats were treated with methotrexate in a dose of 0.3 mg,`kg 24 hr and 1 hr before the carrageenan injection. The degree of cellular infiltration and type of dominant cells was however unchanged. DISCUSSION The role of cellular hypersensitivity in the pathogenesis of adjuvant arthritis has been well demonstrated. Our observations herein suggest that the sensitized lymphoid cells which migrate into the site of the carrageenan-induced inflammation in rats inocu lated with adjuvant act as a trigger to produce the prolonged phase of ACII. The prolonged phase began to occur 1-2 days after the carrageenan injection, which was corresponds roughly to the time of lymphoid cell migration into the inflamed paw. Many immunosuppressive drugs suppress primary immune response and experi mentally induced delayed hypersensitivity in humans as well as in animals. However several investigators have pointed out that treatment with immunosuppressive drugs often improved clinical features without alterating established immune response. Impro vement of symptoms by immunosuppressive drugs in rheumatoid arthritis and systemic lupus erythematosus does not appear to be related to the changes in titer of immunologic reaction or amount of immunoglobulin (3, 4, 5). Thus, immunosuppressive drugs ap pear to have a certain type of anti-inflammatory action in addition to their immunosup pressive effects. 6-Mercaptopurine was found to alter the inflammatory cycle in rabbits , virtually eliminating the participation of mononuclear cells (6). Hersh et al . (7) studied the ef fects of nme!hotre-yate and 6MP on the local inhainnatory response in humans and also observed an inhibition of the mononuclear cell phase. It has also been reported that
the anti-inflammatory effects of 6MP are due to suppression of a bone marrow response to local inflammation (8). In the present study, the prolonged phase of ACII was markedly inhibited by im munosuppressive drugs given shortly before the carrageenan injection. For the follow ing reasons it is unlikely that the inhibition was due to immunosuppressiveeffects of the drugs: 1) The drugs given before 5th (Table 2) or 6th (Table 3) post-inoculation day did not suppress the prolonged inflammation, rather the immunosuppressiveaction was usually more prominent when the drugs had been given shortly after or just before antigen stimulation (9). 2) As reported previously, the prolonged phase was produced on the 5th post-inoculation day when carrageenan was injected on 3rd day (1). In this study, however, immunosuppressive drugs given from 5th to 7th or 8th to 10th post-inocula tion days intensively suppressed the prolonged phase. Since acute inflammation such as thermal injury and turpentine-induced pleurisy was suppressed by 6MP, methotrexate and cyclophosphamide (10), the simple anti-in flammatory action of immunosuppressive drugs was suggested. However carrageenan edema and the early phase of ACII were not suppressed by these drugs in this study. Therefore, the suppression of the prolonged phase of ACII by immunosuppressivedrugs was not considered to be due to the non-specific anti-inflammatory action of the drugs. Moreover methotrexate did not inhibit cellular migration into the inflamed lesion. As discussed above, the mechanism of inhibitory effects of immunosuppressivedrugs on the prolonged phase of ACII remains unclear, however, since lymphoid cells are con sidered to mediate the prolonged phase of ACII and immunosuppressive drugs affect some lymphoid cells, the suppression of the prolonged phase by the drugs appears at tributable to the action of drugs on certain biologic activities of sensitized and,'or non sensitized lymphoid cells which migrate into the rats paw. The effects of these drugs on the release of lymphocytic mediators or rnacrophage-lymphocyte interaction have yet to be determined. It is thus concluded that certain activities of immunosuppressive drugs other than immunosuppressive and simple anti-inflammatory actions play a role in the mechanism of action of these drugs. Acknoltwleci±,enients: Thanks are due to Mr. M. Tsubokawa, Mr. A. Yamada and Mr. M. Kato for skillful technical assistance. REFERENCES 1) MIZUSHIMA, Y., TSUKADA, W. ANDAKIMOTO, T.: Ann. rheum.Dis. 29, 178(1970) 2) VANARMAN, C.G., BEGANY, A.J., MILLER, L.M. ANDPLESS, H.H.: J. Pharmacol.exp. Ther. 150, 328 (1965) 3) ALEPA, F.P., ZVAIFLER, N.J. ANDSLINWINSKI, A.J.: Arthr. and Rheum.13, 754 (1970) 4) SWANSON, M.A., ANDSCHWARTZ, R.S.: New. Engl. J. Med. 277, 163 (1967) 5) DADSON, W.H. ANDBENNETT, J.C.: J. clin. Pharmacol.9, 251 (1969) 6) PAGE,A.R., CONDIE, R.M. ANDGOOD,R.A.: Am. J. Path. 40, 519 (1962) 7) HERSH, E.M., WONG,V.G. ANDFREIREICH, E.J.: Blood,27, 38 (1966) 8) HURD,E.R. ANDZIFF,R.: J. exp. Med.128, 785 (1968) 9) MAKINODAN, T., SANTOS, G.W. ANDQUINN,R.P.: Pharmacol.Rev. 22, 189 (1970) 10) STEVENS, J.E. ANDWILLOUGHBY, D.A.: J. Path. 97, 367 (1969)