Journal canadien
`Canadian Journal
`·or Physiology
`de physiologie
`and Pharmacology et pharmacologie
`
`Puhlish~d by
`THE NATIONAL RESEARCH COUNCIL OF CANADA
`
`Publie par
`LE CONSEIL NATIONAL DE RECHERCHES DU CANADA.
`
`Volume 55 Number I
`
`February 1977
`
`Volume 55
`
`numero 1
`
`fevrier 1977
`
`A Markov chain characterization of human neutrophil locomotion
`under neutral and chemotactic conditions
`
`ABRAHAM BOYARSKY
`Department of Mathematics, Sir George Williams Campus, Concordia University,
`Montreal, P.Q., Canada H3G 1M8
`
`AND
`PETER B. NOBLE1
`Department of Ph ysiology, Faculties of Medicine and Dentistry,
`McGill University, Montreal, P.Q., Canada H3A 2BZ
`Received January 8, 1976
`
`BOYARSKY, A., and NOBLE, P. B. 1976. A Markov chain characterization of human
`·neutrophil locomotion under neutral and chemotactic conditions. Can. J. Physiol. Pharma(cid:173)
`col. 55, 1-6.
`The locomotion of human neutrophils is modelled by a continuous-time Markov chain
`model consisting of five states: state 0, where the cell is stationary, and four motile states
`whose directions are defined by the four quadrants of a Cartesian plane. In this paper,
`the Markov property is verified experimentally in special cases. Further experimental
`evjdence for tlie model is provided by the waiting-time distributions in each of the five
`states, which are well approximated by exponential distributions. Using the steady-state
`distribution of the Markov chain as a measure of the ultimate motion of the cells, it is
`possible to detect the effect of known cbemotactic agents upon neutropbil locomotion.
`Other useful parameters describing neutropbil locomotion are presented .
`
`BOYARSKY, A. et NOBLE, P. B. 1976. A Markov chain characterization of human
`neutrophil locomotion under neutral and chemotactic conditions. Can. J. Physiol. Pharma(cid:173)
`col. 55, 1-6.
`La locomotion des neutropbiles bumains se fait sur un modele represente par une cbaine
`de Markov a temps continu, consistant en cinq etats: etat 0 quand Ia cellule est stationnaire
`et quatre etats mobiles dont les directions sont definies par les quatre quadrants du plan
`Cartesien. Dans cet article, la propriete de Markov est verifiee experimentalement dans
`des cas particuliers. Une preuve experimentale supplementaire en faveur du modele est
`foumie par Jes distributions du temps d'attente dans chacun des cinq etats, bien representees
`par des distributions exponentielles. A !'aide de la distribution d'etat stationnaire de la
`cba\ne de Markov prise comme mesure du mouvement fondamental des cellules, il est
`possible de determiner l'effet d'agents cbemostatiques connus sur la locomotion neutro(cid:173)
`pbile. D'autres parametres utiles dans la description du mouvement des neutrophiles sont
`presentes.
`
`. [Traduit par le journal]
`
`1 All reprint requests should be addressed to P. B. Noble.
`
`=
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`48
`
`Inhibition of the immune response by .
`rapamycin, a new antifungal antibiotic
`
`R. R. MARTEL, J. KLICIUS, AND S. GALET
`Department of Pharmacology, Ayerst Research Laboratories,
`Montreal, P.Q., Canada H4R 116
`Received June 30, 1976
`
`MARTEL, R. R., Kucrus, J., and GALET, S. 1977. Inhibition of the immune response by
`rapamycin, a new antifungal antibiotic. Can. J. Physiol. Pharmacol. 55, 48-51.
`Rapamycin, a new antifungal antibiotic, was found to inhibit the immune response in
`rats. It totally prevented the development of two experimental immunopathies ( experi(cid:173)
`mental allergic encephalomyelitis (EAE.) and adjuvant arthritis (AA)) and the formation
`of humoral (IgE-like) antibody. It was about half as potent as cyclophosphamide in
`inhibiting EAE. In AA and on antibody formation, rapamycin and cyclophosphamide
`were about equipotent, whereas metbotrexate was more potent. The immunosuppressant
`activity of rapamycin appears to be related to inhibition of the lymphatic system.
`
`MARTEL, R. R ., Kucrns, J. et GALET, S. 1977. Inhibition of the immune response by
`rapamycin, a new antifungal antibiotic. Can. J. Physiol. PharmacoL 55, 4~-51.
`La rapamycine, un nouvel antibiotique antifungique, inhibe la reponse immunitaire
`chez le rat. II empeche totalement le developpement de deux immunopathies experi(cid:173)
`mentales (encephalomyelite experimentale allergique (EEA) et arthrite adjuvante (AA))
`et la formation d'anticorps bumoraux semblables aux JgE. Sa puissance est la moitie de
`celle du cyclophospbamide dans l'inhibition de l'EEA. L'effet sur l'AA et sur la formation
`d'anticorps est approximativement le meme pour la rapamycine et le cyclopbosphamide
`alors que le methotrexa1e est plus puissant. L'activite immunosuppressive de la rapamycine
`parait liee a une inhibition du systeme lympbatique.
`
`[f raduit par le journal]
`
`Introduction
`R apamycin is a recently described (Vezina
`et al. 1975; Sehgal et al. 1975) antifungal
`from a
`antibiotic extracted
`streptomycete
`(Streptomyces hygroscopicus) isolated from an
`Easter Island soil sample. It is particularly ef(cid:173)
`fective against Candida albicans both in vitro
`and in vivo (Sidorowicz et al. 1975).
`The inhibitory effects of this new antibiotic
`on two experimental immunopathies (EAE and
`AA) and on the formation of IgE-like antibody
`are r:ies~ribed in this report.
`
`Methods
`
`EAE
`EAE was induced in female, inbred Wister- Lewis
`rats (120-140 g). The rats were injected in the left
`hind foot pad with 0.05 ml. of an emulsion consisting
`of guinea pig spinal cord (4 .2 g) homogenized in a
`mixture of 5.8 ml of 0.5% aqueous phenol and an
`equal volume of complete Freund's adjuvant contain(cid:173)
`ing 4.4 mg/ml of heat-killed, dried Mycobacterium
`
`ABBREVIATIONS: EAE, experimental allergic enceph(cid:173)
`alitis; AA, adjuvant arthritis; po, per os; EA, egg
`albumin; PCA, passive cutaneous anapbylaxis.
`
`butyricum (Difeo). The sensitized rats were treated
`orally with rapamycin or cyclophosphamide according
`to different schedules (see Table 1). The animals
`were observed for signs of hindleg paralysis from day
`I 0 to day 16 (day of sensitization is day 0). The rats
`that did not show paralysis of the bindlegs during the
`observation period were considered protected.
`
`AA
`AA was induced in male inbred Wister-Lewis rats
`( 180-200 g) . The rats were injected intradermally in
`the foot pad of the left hindpaw (day 0) with 0.05 ml
`of a fine suspension of kjlled and dried M. butyricum
`(Difeo) at a concentration of 5 mg/ml in liquid paraffin
`( Freund's adjuvant). For
`the prophylactic study,
`compounds were administered per os daily starting on
`the day of adjuvant injection (day 0) and until day 16.
`For the therapeutic study, treatment was started on
`day 14 and .continued until day 22. Hindleg volume
`was determined by mercury displacement · 2 b after
`·the last treatment. HindJegs were ~ipped in mercury
`up to the hair line. The mercury displaced represents
`the volume of the hindlegs ( 13 .6 g of mercury = 1 ml).
`lgE-like Antibody Formation
`A modification of the method described by Mota
`( 1964) was used to -produce lgE-like antibody in the
`rat. The effect of rapamycin on this response was
`studied. Male Charles River .r:ats ( 180-200 g) were
`injected intraperitoneally with I ml of killed Bordetella
`pertussis cells (2 X 10~ 0 cells/ml) and intramuscularly
`
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`MARTEL ET AL.
`
`TABLE I. Effect of rapamycin oa EAE
`
`49
`
`Treatment,
`daily
`
`Duration,
`d
`
`Dose,
`mg/kg, po
`
`Rats paralyzed4
`
`Rats sensitized
`
`% . protected
`
`Control
`· Rapamycin
`
`Cyclophospharnide
`
`0-13
`0-13
`0-13
`0- 6
`7-13
`0-13
`0-13
`0-13
`
`10
`5
`2.5
`10
`10
`5
`2.5
`1
`
`33/41
`0/12
`9/18
`11/17
`2/12
`7/12
`1/lg
`9/19
`4/6
`
`20
`100
`50
`35
`83
`42
`95
`53
`33
`
`C1RatS which showed hindleg paralysis between day 10 and 16.
`
`with 0.3 ml of EA ( 10 mg/ml). Oral administration
`of rapamycin and other compounds was started at the
`same time (day 0) and continued daily until day 11.
`On day 12, the animals were anesthetized with ether
`and bled from the abdominal aona. Tbe presence of
`EA antibody in tbe serums of control and treated
`groups (eight animals in each group) was determined
`by the PCA method. The pooled undiluted serum (0.1
`ml) of each group was injected intradermally in six
`rats (three sites per rat). Forty-eight hours later the
`animals were challenged intravenously with 11.25 mg
`of EA per kilogram in 1 % Evans blue dye in saline
`(0.9 ml per 100 g). After 30 min the rats were killed
`by CO. inhalation and the diameter of the wheals
`formed oo the underside of the skin was measured.
`The cydophospbamide used in these studies was
`purchased from ICN Pharmaceutical, Inc., and tbe
`methotrexate was generously supplied by Lederle
`Laboratories.
`
`Results
`
`Effect on EAE
`BAE is characterized by paralysis of the hind
`quarter. Rapamycin ( 10 mg/kg, po), admin(cid:173)
`istered daily for 14 d starting on the day of
`sensitization (day 0) completely prevented the
`development of hindleg paralysis (Table 1).
`None of these rats (10 mg/kg) showed late
`paralysis when observed until day 21.
`Rapamycin was more active when administered
`early (day 0 to 6) in the course of EAE than
`when administered from day 7 to 13 (Table 1).
`These data suggest that rapamycin exerts most
`of its effect during the induction of EAE.
`The irnmunosuppressive drug cyclophos(cid:173)
`phamide, a potent inhibitor of EAE (Rosen(cid:173)
`thale et al. 1969), appeared to be about twice
`as potent as raparnycin in 'these experiments.
`
`Effect on AA
`AA is characterized by a severe inflammatory
`
`reaction of the hindlegs. When the adjuvant
`(mycobacteria in oil) is injected into a hindpaw
`(day 0) and treatment started on that day
`(prophylactic treatment), drugs can be evalu(cid:173)
`ated for their effect on two distinct inflammatory
`phases of the disease: (a) and early phase in the
`injected paw which peaks around day 3 and is
`mainly dependent on an acute inflammatory
`reaction to the adjuvant, and ( b) a late phase
`(starting around day 10) in the injected and
`the noninjected hindlegs, resulting from a d~
`layed or cellular-type hypersensitivity reaction
`to some constituent of
`the mycobacteria
`(Rosenthale 197 4) . The immunosuppressive
`agents inhibit only the late or immune phase,
`whereas the anti-infiao:µnatory drugs inhibit
`both phases (Walz et al. 1971; Rosenthale
`1974).
`Rapamycin (5 mg/kg), cyclophosphamide
`(5 mg/kg), and methotrexate (0.25 mg/kg),
`when administered orally, completely block.!}.d
`the secondary immune response (day 16) in
`both hindlegs. However, they did not decrease
`significantly the primary nonhnmune phase in
`the injected paw. The protective effect of the
`three compounds was still complete on day 22,
`6 d after treatment was stopped (Table 2). The
`doses reported in the table are about the lowest
`that will inhibit AA completely.
`In established arthritis (six controls and six
`treated), rapamycin ( 10 mg/kg) was inactive.
`It did not prevent further swelling of the hind(cid:173)
`paws. However, the swelling was slightly less
`than in the untreated arthritic rats.
`
`Effect of lgE-like Antibody Formation
`Twelve days after injection of EA and B.
`pertussis, the pooled serums of control and
`
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`50
`
`CAN.). PHYSIOL. PHARMACOL. VOL. SS, 1977
`
`TABLE 2. Effect ofrapamycin on AA
`
`Hindleg volume, ml ± SE
`
`Injected
`
`Day 16
`
`2.3 ± 0.06
`4.9 ± 0.24
`3.1±0.02
`3.6 ± 0.26
`3.3 ± 0.15
`
`Day 22
`2.4 ± 0.06
`5.7 ± 0. 22
`2.9 ± 0.24
`3.3 ± 0.21
`3.2 ± 0.12
`
`Nooinjected
`
`Day 16
`2.3 ± 0.06
`3.3 ± 0.24
`2.2 ± 0.12
`2.1 ± 0.02
`2.2 ± 0.04
`
`Day 22
`2.4 ± 0.06
`4.2 ± 0.23
`2.3 ± 0.19
`2.2 ± 0.22
`2.2 ± 0.04
`
`Treatment a
`
`Day 3
`2.1 ± 0.02
`Normal control
`3.7 ± 0.08
`Arthritic control
`3.7±0.10
`Rapamycin, S mg/kg
`3.4 ± 0.10
`Cyclophosphamide, 5 mg /kg
`3.9 ± 0.20
`Methotrexate, 0.25 mg /kg
`aorally from day 0 to day 16 (8-10 rats per group).
`
`TABLE 3. Effect of rapamycin on IgE-like
`antibody formation
`
`Discussion
`
`Rapamycin, a new antifungal antibiotic, has
`been found to inhibit the immune response.
`Small, well-tolerated doses, 5-10 mg/kg,
`(Sidorowicz et al. 1975) of this antibiotic totally
`prevented the development of cellular immunity
`(EAE and AA), as well as the formation of
`IgE-like antibody. In BAE, rapamycin appeared
`to be half as potent as cyclophosphamide.
`In AA and antibody formation, the potency of
`rapamycin and cyclophosphamide appeared
`comparable, whereas rnethotrexate was more
`potent. Similarly, Walz et al. (1971) used 20-
`tirnes Jess methotrexate than cyclophosphamide
`to block AA, and Rosenthale et al. ( 1969)
`showed methotrexate to be much more potent
`than cyclophosphamide in inhibiting paralysis
`in EAE.
`All evidence indicates that the inhibitory
`effect of rapamycin in EAE and AA depends
`on suppression of
`the
`immune response.
`Rapamycin produced a complete and long(cid:173)
`lasting inhibition of BAE. Prophylactic treat(cid:173)
`ment with rapamycin in the AA model in(cid:173)
`hibited only the immune-mediated phase of
`inflammation and the inhibition lasted after
`treatment was discontinued. In established AA,
`10 mg of rapamycin per kilogram, a dose
`which completely prevented the development
`of EAE and AA, was inactive. This profile was
`reported for immunosuppressive agents such
`as azathioprine, methotrexate, cycloleucine,
`and cyclophospbamide by Rosenthale (197 4).
`Nonsteroidal anti-inflammatory drugs do not
`produce fuII protection in EAE (Komarek and
`Dietrich 1971 ) . In AA, the steroidal and non(cid:173)
`steroidal anti-inflammatory drugs inhibit both
`the early nonspecific and the late immiine
`
`Treatment,
`daily
`
`Dose,
`mg/kg, po
`
`Control
`Rapamycin
`
`Cyclophosphamide
`
`10
`3
`10
`3
`0.3
`25
`
`Methotrexate
`Phenylbutazone
`ap < 0.01.
`Non:: The skin was sensitized with 0.1 ml of the pooled serum of
`eight rats per group.
`
`Diameter of
`skin wheal,
`mm± SE
`28 .1 ± 0.8
`0
`10.2 ± 0.24
`0
`12.0 ± 0.44
`0
`27.0 ± 0.8
`
`treated rats were assayed for the presence of
`... EA antibodies by the 48-h PCA method. As
`shown in Table 3, the serum of the rats treated
`with 10 mg of rapamycin per kilogram and
`cyclophosphamide produced no skin wheals.
`A smaller dose of rapamycin and cyclophospha(cid:173)
`mide partially inhibited the response, while
`methotrexate was more potent. Rapamycin
`(100 mg/kg, po, administered to sensitized rats
`1 h previous to challenge) or diluted an~i-EA
`serum (one part with three parts of serum ob(cid:173)
`tained from unsensitized rats . treated with
`rapamycin, 50 mg/kg orally for 2 d) had no
`effect on the PCA response. From these results
`it was concluded that rapamycin, similar to the
`immunosuppressive agents cyclophospham.ide
`and methotrexate, inhibited antibody forma(cid:173)
`tion. The nonsteroidal anti-inflammatory drug
`phenylbutazone had no effect. No drug-related
`adverse effects could be noted, apart from a
`depression of the growth curve with rapamycin
`and the other immunosuppressive agerits.
`
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`MARTEL ET AL
`
`Sl
`
`phases of inflammation (Walz et al. 1971; Wax
`-et al. 1974). They are about equipotent when
`assayed by the preventive and the therapeutic
`methods (Perper et al. 1971; Walz et al. 1971;
`wax et al. 1974; Martel and Klicius 1976).
`Arthritis occurs readily after dosing is stopped
`(Perper et al. 1971 ) . Furthermore, i:apamycin
`inhibited antibody formation, whereas the non(cid:173)
`steroidal anti-inflammatory drug phenylbuta(cid:173)
`zone did not.
`The mechanism of action of rapamycin on
`the immune system is unknown at the present
`time. However, long-term toxicity studies in
`dogs (Hemm, R. D., and Authier, L., personal
`communication)
`have demonstrated
`that
`rapamycin caused hypoplasia of lymphatic tis(cid:173)
`sues (lymph nodes, spleen, thymus). Thus, it
`·appears that the activity of this antibiotic on
`the immune response depends on an inhibition
`of the lympathic system.
`
`Acknowledgments
`The authors gratefully acknowledge the able
`technical assistance of Mrs. Lise Brisebois and
`Miss Josee Caron.
`
`KOMAREK, A., and DIETRICH. F. M. 1971. Chemical
`prevention of experimental allergic encephalomye(cid:173)
`litis in rats: a quantitative evaluation of steroids
`and various non-steroid drugs. Arch. Int. Pharma(cid:173)
`codyn. 193, 249-257.
`MARTEL, R. R., and Kucrus. J. 1976. Anti-inflamma(cid:173)
`tory and analgesic propertie:S of etodolic acid in rats.
`Can. J. Pbysiol. Pharmacol. 54, 245-248.
`
`MoTA, I. 1964. The mechanism 'Of anaphylaxis. I.
`Production and biological properties of mast ceU(cid:173)
`sensitizing antibody. Immunology, 7, 681-699 .
`PERPER, J. R., ALVA.REX, B., CotoMBo, c., and
`SCHRODER, H. I 971. The use of a standardized
`adjuvant arthritis assay
`to differentiate between
`anti-inflammatory and · irnmunosuppressive agents.
`Proc. Soc. Exp. Biol. Med.137, 506-512.
`RosENTHALE, M . E. 1974. Evaluation for immuno(cid:173)
`suppressive and antiallergic activity. In Anti-inflam(cid:173)
`matory agents, chemistry and pharmacology. Vol.
`II. Edited by R. A. Scherrer and M. W. Withehouse.
`Academic Press Inc .. New York. pp. 123-192.
`RosENTHALE, M. E., DATKO, L. J., K.AssARICH, J., and
`SCHNEIDER, F. 1969. Chemotherapy of experimental
`Int.
`allergic
`encepbalomyelitis
`(EAE). Arch.
`Pharmacodyn.179, 251-275.
`SEHGAL, X. N., BAKER, H., and VEzINA, C. 1975.
`Rapamycin (AY-22,989), a new antifungal anti(cid:173)
`biotic. II. Fermentaticn, isolation and characteriza(cid:173)
`tion.]. Antibiot. 28, 727-732.
`SIDOROWICZ, H., BAKER, H., and V.EzINA, C. 1975.
`Rapamycin (AY-22,989), a new antifungal anti(cid:173)
`biotic.: in vitro and in vivo studies. 15th Interscience
`Conference on antimicrobial agents and chemo(cid:173)
`therapy, abstract 26.
`VEZINA, C., KunEtsKr, A., and SEHGAL, S. N. 1975.
`Rapamycin (A Y-22,989), a new antifungal anti(cid:173)
`biotic. I. Taxonomy of the producing streptomycete
`and isolation of the active principle. J. Antibiot. 28,
`721-726.
`WALZ, D. T., DIMARTINO, M. J., and MrsHER, A. 1971.
`Adjuvant-induced arthritis· in rats. II. Drug effects
`on physiologic, biochemical and
`immunologic
`parameters. J. Pharmacol. Exp. Ther. 179, 223-231.
`WAX, J., WINDER, C. V., TESSMAN, D. K., and
`STEPHENS, M. D. 1974 Comparative activities, toler(cid:173)
`ances and safety of non-steroidal anti-inflammatory
`agents in rats. J. Phann. Exp. Ther. 192, 172-178.
`
`SJ
`
`= us
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