EXPERIMENTAL

AND

Effects

MOLECULAR

PATHOLOGY

103-112

(1977)

of Cycloleucine on Macrophages Allergic Encephalomyelitisl SEY~~OUR LEVINE

Pathology

26,

Department,

AND

New York Medical Hospital,

College Island, New

Roosevelt

Received

July

and

on Experimental

RICHARD SOWINS~I Center for York, New

Chronic Disease, York 10044

Bird

S. Coler

8, 1976

Cycloleucine is known to suppress the development of experimental allergic encephalomyelitis (EAE ), an autoimmune disease produced by immunization with central nervous system (CNS) antigen and manifested by inflammation in the CNS. The present work shows that cycloleucine inhibited EAE produced by passive transfer of cells from fully immunized donor rats. The drug also inhibited macrophage response in simplified models for the inflammatory events at sites of antigen inoculation and in target neural tissues. However, cycloleucine was not completely specific for macrophages. The data suggest that cycloleucine inhibits both the early antigenprocessing stage and the late lesion-development stage of EAE, and that the macrophage is an important but not unique target cell. Inhibition was not due to interference with transport of inoculum into draining lymph nodes or to activation of the adrenal cortex.

INTRODUCTION Cycloleucine ( 1-aminocyclopentane carboxylic acid) is an immunosuppressive drug with activity against experimental allergic encephalomyelitis (EAE), adjuvant arthritis, homograft rejection and antibody production (Rosenthale et al., 1972). The kinetics and mechanism of its action are complex and incompletely known. There is evidence that cycloleucine interferes with processing of antigens in macrophages ( Frisch and Wilson, 1969). One way that a drug can interfere with processing is for the drug to prevent the accumulation and multiplication of macrophages at the site of antigen deposition. The present study is a direct approach to this problem by histologic evaluation of macrophage response at sites of tissue necrosis or injection of foreign material. Also, the mechanism of druginduced immunosuppression has been elucidated by its effects on the passive transfer of EAE, a method which permits dissection of immunizing events in the donor rats from lesion formation in the recipient animals. METHODS Male Lewis rats from Microbiological Associates, Inc., were maintained in hanging wire cages on Purina Laboratory Chow and tap water. Cycloleucine was dissolved in saline at 15 mgm/ml. It was administered by gastric tube with1 Supported by a grant from the Kroc Foundation. Cycloleucine kindly supplied by Dr. bI. E. Rosenthale, Wyeth Laboratories. Photomicrographs by XI. Moritz.

Copyright All rights

@ 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0014-4800

104

LEVINE

AND SOWINSKI

out anesthesia. Ether anesthesia was used for surgery, inoculations, and exsanguination. Adrenalectomies were done through a dorsal midline incision. Rats were maintained on 0.9% NaCl as sole fluid thereafter. Macrophage accumulation was studied in response to focal brain necrosis and at sites of injection of brain homogenate, Freund’s incomplete adjuvant or spinal cord-complete adjuvant emulsions. Closed brain injuries were produced by incising the scalp and applying one of the faces of a pyramid-tip preheated 379 watt electric soldering iron to the intact skull for 7 seconds. The heat produced an oval zone of necrosis occupying the posterior dorsal surface of the right cerebral hemisphere. The lesion extended through the cerebral cortex into underlying white matter and hippocampus. The scalp incision was closed with clips. Some of the rats also received 0.1 ml of a 10% rat brain homogenate at each of two subcutaneous sites in the right flank, and 0.1 ml of incomplete Freund’s adjuvant (IFA) at two similar sites on the left side. The homogenate was prepared by straining fresh brain through a stainless steel sieve, heating to 60°C for 1 hour, adding saline and cycling between two syringes. The adjuvant consisted of 85 parts of Bayol F mineral oil and 15 parts Arlacel A. Other rats were given 0.05 ml of an emulsion prepared with equal parts of 40% guinea pig spinal cord homogenate and complete Freund’s adjuvant (CFA = IFA plus 4 mgm/ml killed tubercle bacilli). The emulsion was injected into one of the pads on the sole of the right foot. Three or 7 days later, the rats were exsanguinated. Spleen and thymus were weighed fresh. Brains and lymphoid organs were fixed in Bouin’s fluid, flank skin in formalin, and right feet and vertebrae in picric acid-formic acid-formalin. Frontal slices of brain and slices of other tissues were embedded in paraffin, cut and stained with hematoxylin-eosin. For passive transfer experiments, EAE was produced by active immunization with guinea pig spinal cord emulsified in CFA in the foot pad and pertussis vaccine in the dorsum of the same foot. These donor rats were killed when they had clinical signs of EAE. Their lymph nodes draining sites of inoculation were removed and processed into a viable cell suspension (Levine and Hoenig, 1968). These “EAE cells” were injected into normal male Lewis recipients. The recipients were checked daily for signs of EAE. After 8 or 9 days, their spinal cords were removed and studied for histologic lesions. RESULTS Passive Transfer of EAE It is known that cycloleucine inhibits the development of EAE after immunization of rats with CNS antigen and adjuvants (Rosenthale et al., 1972). The drug might inhibit the inflammatory and immunologic events at the site of inoculation, in the draining lymph node, or in the target CNS tissue. The passive transfer system with cycloleucine treatment of recipient rats only permitted the separate evaluation of drug effects on the interaction between fully sensitized lymphoid cells and the target neural tissue. Control, nondrugged rats that received EAE cells from pooled donors developed clinical signs of EAE 4 or 5 days after transfer of donor lymph node cells,

CYCLOLEUCINE

INHIBITS

MACROPHAGES

TABLE

AND

105

EAE

I

Cycloleucine Suppresses EAE in Recipients of Passive Transfer Cycloleucine Day” -8 -4 0 0 +4

EAE signs

mgm/km

EAE Lesions”

Incidence*

Onsetc

Severityd

g/g l/4 l/8 4/8 O/4 4/4

4.8 7.0 6.0 3.0 4.3

2..5 1.0 1.o 0.8 0 2.8

None I,50 1.50 150 300 LjO

3.3 2.7 2.0 1.8 0 2.8

” Days before (-) or after (+) EAE cell t,ransfer. Nondrugged rats were given saline. b Numerator, number of rats with signs. Denominator, total number of rats. Two experiments are combined. c Average number of days after cell transfer. d Paralysis = 3, weakness = 2, flaccid tail = 1. Only those with signs were averaged. e Scored from 4 to zero, based on number of lesions in sections of entire spinal cord. All scores were averaged.

The signs progressed from tail weakness to limb weakness to paralysis in almost every rat. Paralysis lasted only 1 to 3 days, so that the rats were partly recovered at the time of sacrifice, 8 or 9 days after cell transfer. Despite clinical improvement, histologic study revealed numerous perivascular inflammatory EAE lesions in the spinal cord. Administration of a single 150 mgm/kgm dose of cycloleucine to cell recipients on the day of cell transfer or several days before, reduced the incidence of TABLE II Schedules of Cycloleucine Treatments, and Their Effects on Spleen and Thymus No. of Rats

Cycloleucinea Days

3 3 6 3 6 9 3 9 9 9

-4 0 -4, 0 -4 None -4 0 -4, 0 -4, 0 None

Adxc

mgm/kgm 1.50 1.50 150 x 2 300 None 150 150 75 x 2 130 X 2 None

Weight loss (‘% of control)b Spleen

D-4 D-4 D-4 D - 4 D-4

8 8 4 4

12 13 9 14 -

Thymus 33 26 43 39 37 24 29 49 -

~1Day zero = day of thermal brain injury and subcutaneous injections. Controls were given saline orally. Five experiments are summarized. b Rats sacrificed Day + 3. Body size varied among the experiments, and cycloleucine caused loss of body weight; therefore, organ weights were first converted to y0 of body weight and the weight loss was calculated in reference to the 70 of body weight for organs from untreated control groups. c Bilaterally adrenalectomized Day -4, maintained on saline.

106

LEVINE

AND SOWINSKI

FIG. 1. Dorsolateral margin of 3-day-old thermal brain injury, just under pia mater of a control rat. Rounded, engorged macrophages are closely packed. This field was selected to illustrate maximal reactivity in this animal’s lesion. (H&E, X686) clinical signs (Table I). The few signs that developed were delayed in onset and mild (no paralysis). Similar results have been obtained by others (L. Levy, personal communication). Histologic lesions were also reduced. A larger dose (300 mgmfkg m ) e1iminated all signs and lesions. Treatment delayed until the onset of signs did not prevent progression or reduce lesions.

Macrophage Response in Brain Inhibition of passive transfer indicated that cycloleucine had an effect on the interaction of EAE cells and target tissue and/or an effect on the production of lesions. Lesions of EAE contain a complex mixture of mononuclear cells. Therefore, the drug was studied in thermal brain lesions which have an almost pure population of macrophages. Three days after injury, control rats had an abundance of macrophages in the periphery of the necrotic brain tissue, as has been described (Levine, 1971; Levine and Hoenig, 1968). The number of macrophages varied at different sites and at different cross-sectional levels. The response was along the perimeter usually maximal in the subpial part of the perimeter where macrophages were frequently densely crowded and too numerous to count (Fig. 1). The injury caused a mild vascular hyperplasia accompanied by very few lymphocytes or granulocytes.

CYCLOLEUCINE

INHIBITS

I’ See Table I. * average number of days after ino~r~lation c I.50 mgm/kgm orally on day of inoc~llxti~rn.

MACKOPHAGES

(nit11

range

AND

EAE

107

in parentheses).

Rats given a total dose of 150 mgm/kgm cycloleucine according to any of several schedules (Table II) had a reduced influx of macrophages at 3 days. At no point along the perimeter were the macrophages crowded. In five experiments, randomized slides read “blind” were correctly sorted into treated and control categories. In three of the trials, all the rats had been adrenalectomized 4 days before injury, but the results were the same. Rats that were given a total dose of

ITI

Effects of cycloleucine on macrophages and on experimental allergic encephalomyelitis.

EXPERIMENTAL AND Effects MOLECULAR PATHOLOGY 103-112 (1977) of Cycloleucine on Macrophages Allergic Encephalomyelitisl SEY~~OUR LEVINE Patholo...
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