The effects of polyinosinic: Polycytidylic acid (pl:C) on the GVH reaction: Immunopathological observations

CLINICAL

IMMUNOLOG>

,\NI)

IMML’NOYAI

HOLOGY

39.

i()?-

1 1 i I i’)Xhl

The Effects of Polyinosinic: Polycytidylic GVH Reaction: lmmunopathological

The reaction

effect\ were

of polyinosinic: studied. The

men& rental

natural lymphoid

killer tNK) cell cells intravenously

reaction red blood

wah monitored cell5 tSRBC)

with

pl:C

prior

with pl:C had logical changes. NK

cells.

activity. into

the arnt’t-Lcr-\us-host induce5 interferon

GVH reactions were induced by F, hyhrid mice. The development

BhAF, post-GVH

recipients developed profound In addition. pathological

Mere gland

mice. the induction.

cell (PFC) When 30

:

(GVHI and auginjecting paof a GVH

re\ponhe IOh 86

cell

transfer

as measured the histological

of regulating

markedI) by the lesions

reduced

the

degree

to ,heep lymphoid

immunosupchanges indica-

seen in the qleen. lymph nodea. thymus. liver. of these mice. However. the treatment of BhAF,

no effect in preventing Thi\ stud! suggests

is capable

acid (pl:C) on and markedly

plaque-forming examination.

to parental

the immune response. failed to demonstrate

polycytidylic pl:C rapidly

by measuring the and bq histological

cells were injected into pression by IO- I? days tive of GVH reaction\ cream. and salivary

drug

Acid (pl:C) on the Observations’

lung. panrecipients

of suppression

PFC response to SRBC. :\lso, such of GVH disease. Treatment of donor

either CiVH-induced that a pl:C-induced the GVH

immunosupprezsion mechanism. possibly I’M \i.dcinlL l’lC\\, IIlL

reacticjn.

of mice mice

or pathoinvolving

INTRODUCTION Graft-versus-host (GVH) disease is a frequent sequela of bone marrow (BM) transplantation. which has become an accepted treatment for aplastic anemia, acute leukemia. and genetically determined immunodeficiency diseases. Experimentally, GVH reactions can be induced by inoculating F, hybrid mice with parental lymphoid cells. In principle. the F, hybrid does not recognize the parental cells as foreign. Conversely. the grafted parental strain cells can recognize and react against antigens derived from the other parental strain on the hybrid’s cells. Experimental GVH reactions closely mimic those of BM transplantation-induced GVH disease in man. i.e., damage to diverse epithelial tissues (I -41, and generalized immunosuppression (5-g). While it is widely presumed that tissue injury in the GVH reaction is inflicted by parental T cells, no direct evidence for this has been provided. Recently, natural killer (NK) cells have been implicated as playing a role in both experimental and human GVH disease. NK cells are endogenously active 1 This Research

work was supported Council of Canada.

1 Supported by should be addressed: treal.

Quebec.

by grants

from

the

a Medical Research Council Department of Physiology.

Canada

H3G

IYh.

National of

Cancer

Institute

Canada studentship. McGill University.

of Canada. XI 3655

whom Drummond

and

the

Medical

correspondence Street.

Mon-

EFFECTS

OF

pl:C

ON

THE

GVH

REACTION

103

killer cells capable of lysing neoplastic and virally infected cells (9), as well as certain types of normal cells (10) irz \qitro. In mice, NK cell activity has been found to be increased (1 I), decreased (12), or both (13, 14) after GVH induction. In a previous study, we have shown that NK cell activity increases early after GVH induction, peaks, and then decreases to levels markedly below normal (13). The same pattern of NK cell activity has been reported in GVH reactions in rats (15). Early, rapid activation of NK cell activity in human BM transplant recipients who develop GVH disease has also been reported (16). Thus, similar patterns of NK cell activity after GVH reactions have been reported in mice, rats. and humans. It has been suggested that NK cells may be the effector cells (13). oi alternately, the target cell (17) during the GVH reaction. However, NK cells have also been implicated in resistance to grafts of both BM (12) and allogeneic lymphoid cells (18). The purpose of this study was to examine the effect of an inducer of interferon (IF) and NK cells on the GVH reaction. The results show that pretreatment of recipient mice with pl:C has a profound effect on the intensity of the GVH reaction. MATERIALS AND METHODS Micr. The parental strains A. C57BLi6 (B6), DBA/? (D2) and their F, hybrids. B6AF, and B6D2F, were used. D2 mice were purchased from the Jackson Laboratory (Bar Harbor, Me.). All other mice were bred in our laboratory. Recipients and donors were 2-3 months old when used. Indrrt~ic~n of’GVH reuctims. Donor spleen and lymph node (inguinal, brachial. axillary, and cervical) cell suspensions were prepared as previously described (19). Cell suspensions were prepared in Hanks’ balanced salt solution (HBSS) by gently passing them through a 50-mesh stainless-steel screen, washing once, and finally passing them through a single layer of cotton gauze. The number of viable lymphocytes was determined using the trypan blue dye exclusion method. The cell suspensions were made up to the desired concentration and passed through gauze a second time. The desired number of viable lymphocytes in a volume of 0.3 ml were injected into the lateral tail vain of each mouse. The dosages used in each experiment are indicated in the Results section. In experiments using B6 donors. the dose used was the lowest which induced profound. long-lasting immunosuppression. Trrcltrnent Lrith polyinosinic.:poI~(,~tidvlic, ucid (pf:C). pl:C (Sigma, St. Louis. MO.) was prepared in HBSS. Mice received two 100 kg injections of pl:C, intraperitoneally. at 18-24 hr and 2-4 hr prior to GVH induction. In experiments where donor mice were treated, pI:C was similarly administered at 18-24 and 2-4 hr prior to sacrifice. pI:C control mice received two injections of pI:C but no parental strain cells. Direct plaque fbrrning cell (PFC) tr.s.say to sheep erytlwoc~tcs (SRBC). Mice were immunized intravenously with 5 x 108 SRBC in aliquots of 0.3 ml. Four days later. spleens were assayed for the total number of direct PFCs to SRBC using the technique described by Cunningham and Szenberg (20) as modified in this laboratory (2 I ).

104

I'EKHS, SEEMAYER.

AND LAPI'

Histology. Animals were autopsied and tissues were fixed in 5/J Formalin. processed for light microscopy, stained with Hematoxylin-phloxine-saffron (HPS), and examined as coded specimens. Tissue\ examined included: thymus. lymph nodes, spleen. parotid, liver, pancreas. and lung. Sttrtistic,.s. P values were dctcrmined by the Student’\ I test.

RESULTS In the first series of experiments. we investigated the effect of pl:C treatment of F, recipients on the degree of GVH-induced immunosuppression. We and others have previously shown that the GVH reaction strongly suppresses both the humoral and cellular (S-8) immune response. Variations in the degree of suppression are indicative of changes in the intensity of the GVH reaction. The rcsuits are presented in Table I. Normal mice produced a vigorous PFC response to SRBC and treatment with pI:C alone had no significant effect on the PFC response. The PFC response of B6AF, hybrid mice treated with 30 x IO” B6 lymphoid cells was reduced to less than YO of the response of normal mice. In contrast, the PFC response of B6AF, mice treated with pl:C prior to parental cell transfer was 40-60% of the PFC response of normal mice. Thus. the pI:C trcament protected the F, recipient mice from GVH-induced immunoauppression. Protection with pl:C was also seen when 40 s 10’1 B6 lymphoid cells were injected into B6D?F, recipients (Table I). However, when A strain donor cells were injected into B6AF, recipients, no pl:C mediated protection could be dcmonstrated, at all cell dosages attempted (Table I ). lmmunosuppression during the GVH reaction undoubtedly involves a complex series of events. It is possible that pl:C interferes with one or more of the steps involved in the generation of such suppression. If so, prevention of suppression may not indicate protection from the GVH reaction itself. If this were true. then one would still expect to see GVH-induced pathological lesions in F, recipients treated with pI:C. Histological studies of pl:C treated and untreated B6AF, mice IS-21 days after induction of GVH reactions with 30 x IOh B6 lymphocytes demonstrate that this is not the case (Figs. l-3 and Table 2). Twelve control F, hybrids were examined. No pathological alterations were recognized. Eleven F, hybrids treated only with pI:C were examined. No pathological alterations were recognized. Fifteen F, animals experiencing GVH reactions were examined from I5 to 21 days post-GVH induction. Variable degrees of thymic dysplasia, nodal and splenic immunoblastic hyperplasia. histiocytic proliferation, and lymphoid depletion with incipient fibrosis. and/or mononuclear cellular infiltrates (hepatic. pancreatic, parotid, pulmonary) were noted in I I mice. The intensity of the alterations varied from slight to severe. In four mice no GVH-associated lesions were recognized. Fifteen GVH-reactive mice which were pretreated with pI:C were examined 13-21 days post-GVH induction. All mice were found to have histologically normal tissues. Thus. treatment of F, recipients with pI:C prior to induction of GVH reactions decreases the immunosuppression and prevents the histological changes induced by the GVH reaction. Next, we investigated the effect of pl:C treatment of donor mice on their ability

EFFECTS OF pI:C ON THE GVH REACTION EFCECT OF

pl:C TREATMENTOF

Parental cell dose and GVH combination 30 ’ IO’>

B6-

Y

TABLE I MKE OYGVH-1~1)uctn I~~ILNOSCPPRESSION”

RKWIENT

Days after GVH induction

B6AF,

105

Experimental ww

PFCispleen

No, of

T t SE x IO 1

N N + pl:C GVH GVH + pl:C

108 x5.9 I.11 45.6

i- 39.x k 0.77 t 0.6X* -+ I I.9=x

N N + pl:C GVH GVH + pl:C

I’4 2 Y8.Y i 0.22 i71.3 2

% Normal

X.53 22.9 0.x!” 27..

100 80 I<11 sx

63 k II.7 1.2 t 0.6” 71 +- II.1

IO0 1 33

30 < IOh Bh - B6AF,

I6

40 x 106 Bh + B6D’iJ F,

I4

N GVH GVH + pl:C

20 x IO” A-t BhAF,

I5

N N + pl:C GVH GVH + pI:C

x5.3 96.3 0 0

16.6 3.0 0” o*

100 II3 0 0

IO X IO” A + B6AF,

13

N GVH GVH + pl:C

91.7 t 10.3 0.31 +- 0.14* 0. IS f 0.09*

100 (
5 y IO”

I5

N N + pl:C GVH GVH + pl:C

X8.1 102 40.3 47.2

IO0 II6 46 54

.4 -+ B6AF,

I f 2 I

iI r t

16.3 21.6 6.0’ 7.x

-

100 77 I ‘ll

ci Each experiment was performed at least twice. giving similar results. The results of one representative experiment are presented. * Significantly different from normal5 (P < 0.05). ** Significantly different from GVH (P i 0.05).

to induce vigorous GVH reactions. pI:C is a potent IF inducer. IF has been shown to have antiproliferative effects in a wide variety of systems [reviewed in Ref. (22)]. Most significantly, IF pretreatment has been shown to inhibit lymphocyte functions both irz vitro (23-25) and in rive (26). Therefore, if pI:C were simply blocking donor cell proliferation and the generation of parental anti-F, cytotoxic T lymphocytes (CTLs). one might anticipate a reduction in the intensity of the GVH reaction if the donor and not the host were treated with pI:C. However, treatment of donor mice with pI:C had no demonstrable effect on the ability of donor cells to induce suppression in F, hybrid mice (Table 3). In addition, no histological differences were noted between GVH reactions induced with pI:C treated donors and untreated donors (data not shown). It would thus appear that the effect of pI:C treatment is not related to the antiproliferative effects of IF.

IO6

f’I:Kf-S.

SL’EMA\I’FK,

.\>I)

I Af’f’

EFFECTS

FIG. 7.

Photomicrographs

( x 100)

< 10” B6 lymphocytes. and histocytic proliferation. tion

of parental

cell\

Lymph

OF

pl:C

of lymph

ON

THE

nodes

from

GVH

a B6AF,

node from a GVH mouse tat illu\trating Lymph node from a GVH mouse which

th) demonstrating

a normal

107

KEACTION

mice

I6 days

after

injection

profound lymphoid received pl:C prior

of 30

depletion to the injec-

architecture.

DISCUSSION The purpose of this study was to examine the effects of pI:C. an agent known to induce IF and NK cell activity, on the intensity of a CVH reaction. The data demonstrate that treatment of F, recipient mice with pI:C sharply decreased the intensity of the GVH reaction, as evidenced by a decrease in immunosuppression and by a lack of histological changes characteristic of the GVH reaction; in contrast, donor treatment with pl:C had no effect on either variable. Also, pl:C induced protection from immunosuppression produced by the GVH reaction was only seen when B6 and not A strain donors were used. Three mechanisms could account for these findings. IF inhibits the suppressor effects of macrophages (77) and T cells (28) in some systems. It is possible that the decreased immunosuppression seen in pt:C

FIG. 3. 30 x IW infiltrate.

Photomicrograph B6 lymphocytes. Salivary gland

phocyte\

(hr.

which

( x 200) of salivary glands from BhAF, mice I6 days after the injection of Salivary gland from a GVH mouse la). illustrating an intense lymphoid from a GVH mouse treated with pI:C prior to the injection of parental lym-

i\; devoid

of any

infiltrate.

Organ

pl:(’

trcatmenl

Histological

in,jury

None

t~requrncy~‘li)t;~l~ Moderate-severe’

Slight-mild,

4 Thymua

I5

Lymph

nodes

Pancreas

I7 I3 17

+

11 The tion

data

represent

results

pooled

of 30 r IO6 B6 lymphocytes h No histological changes were I In thymus.

slight-mild

loss of corticomedullary erate-severe lesions sall’s were

corpuscles, characterized

In lymph nodes with extensive

lesions

were

from

different

were

demarcation characterized

characterked and no or minor by disappearance

moderate-severe by M@ with variable

mild clear

lesions cells.

were characterired whereas moderate-severe

cells

in the

infiltrates

with

by focal

involvement

control

lesion< degrees

L-21

of cortical

damage to meduilary of corticomedullary

or all of the

after

lymphocytes epithelial demarcation

lymph nodes by histiocytes.

a marked and widespread of fibrosis was noted.

infiltrates distinguished

days

the

injec-

mice.

remaining. In the and replacement

periductular lesions were in most

groups

by depletion

with few. if any. epithelial cells by focalized loss of lymphocytes demonstrating replacement

experimental

into BOAF, mice. noted in normal or pl:c‘

slight-mild without

without cells. Modand Haschanges fibrosis.

loss of lymphocytes In the pancreas slight-

by few or modest numbers of mononuby massive numbers of mononuclear ducts.

treated GVH mice was due to IF production which prevents the profound immunosuppression normally produced during a GVH reaction. One might then expect increased pathological injury in pI:C-treated GVH mice since. as we have previously suggested, the immunosuppression generated during the GVH reaction may partially protect the host by also suppressing the donor mediated destructive mechanism(s) (29). However, in pi:C pretreated F, recipients, tissue changes characteristic of the GVH reaction were not seen at all, suggesting that pI:C treatment of F, recipients led to the overall prevention of the GVH reaction. A second possibility is that pI:C. likely by inducing IF production, either directly, or via secondarily induced suppressor mechanisms regulates the parental anti-F, response. IF has been shown to inhibit cellular proliferation (22) and various lymphocyte functions, both in ilitro (23-25) and in \~ivo (26). IF has also been demonstrated to induce suppressor T cells (30). Our observation that pI:C treatment of donor mice had no effect on the GVH reaction suggests that the alteration in the GVH reaction is not due to a direct suppressor effect on donor cells. For a suppressor mechanism to account for all the observations it would be necessary that the suppression: (a) be mediated by the host, rather than the

109

EFFECTS OF pI:C ON THE GVH REACTION TABLE 3 EFFECTOF pl:C TREATMENTOFDONORSONGVH-INDUCED IMMUNOSUPPRESSION”,~ Experimental group N GVH GVH + pl:C (donors treated) GVH + pl:C (recipients treated)

PFCispleen No. of mice 7 3 3

(4 t SE - x IO-‘) 199.2 59.7 + 14.47 59.1 + 71.9”

3

143.4 + 5.7”*

R Normal IO0 30 30 77

‘130 x IOh B6 lymphoid cells were injected into B6AF, recipients. PFC responses were determined 14 days after GVH induction. h Three experiments were performed. All gave similar results. * Significantly different from normal (P < 0.02). ** Significantly different from GVH (P i 0.02.

donor; (b) be specific for suppressing the donor antirecipient response, but not the subsequent host anti-SRBC response; and, (c) be specific for B6 and not A strain parental cells. However, it is difficult to reconcile the required specificity of suppression to B6, but not to A, with the nonspecific nature of the inducing drug, pI:C. While this type of suppression remains a possibility, to our knowledge no evidence to support such a mechanism has been reported. It therefore appears unlikely that the effects of pI:C are due to specific suppression of the parent anti-F, response. The third possibility is that pI:C pretreatment induces a mechanism which rejects some part or all of the parental cell graft. A decrease in the number of surviving transferred parental lymphocytes would be equivalent to decreasing the parental cell dose. In the strain combination used (B6 donors and B6AF, recipients) the administered dose of 30 x 106 lymphocytes was the lowest which produced profound suppression and histological lesions. If 20 x 10” B6 lymphocytes are given. only partial suppression with no pathological changes are observed (data not shown), closely resembling the F, mice grafted with 30 x 106 lymphocytes and treated with pI:C. Rejection of parental strain cells by F, hybrids contradicts the classical laws of transplantation, but such has been observed for parental BM grafts (31) and tumor cells (32. 33). but only when the transferred cells are homozygous H-2b at the D end of the H-2 complex. Sensitivity to rejection is also coded for at a non-H-2 locus. The genes coding for the proposed target antigen have been called’Hh, and appear to be inherited non-codominantly. such that the F, hybrid does not express the gene, whereas the parental strain does (31). This phenomenon has been termed hybrid resistance (HR), and rejection seems to be effected by NK cells (12). HR to BM grafts can be augmented (12, 34. 35) and decreased (12, 34, 36) by agents which augment and decrease NK cell activity, respectively. In addition, HR has been shown to occur against grafts of mature parental lymphoid cells as well (37, 38), but the involvement of NK cells in the rejection of mature resting lymphocytes has not been demonstrated. One difficulty with the concept that NK cells reject the parental cell graft is that

wherea BM cells have been shown to be sensitive to NK-mediated cytotoxicit\ in ~‘itl.o. mature lymphocytes have not ( IO). Phytohemagglutinin (PHA) blast\ i\r< sensitive to natural killing (39. 40). It ma) he that parental Iymphocytc~ onc‘c activated become targets for NK cell\. and. in the c‘;~\c of the pl:c‘ treated rccipients. are rejected mot-c efficiently, accounting tilt- the observed dccreascd intcnsity of the GVH reaction. We have noted ;I marked decrease in the number 01‘ donor lymphocyte4 in F, hybrid mice 2 days after cell transfer. This rapid elimination of donor cells correlates well with incrca\ed Icvels ot’ NK cell activity. No increase in macrophagc tumoricidal activity w;t\ observed (A. Per-e\. F, I? Nestel. T. A. Seemayer. and W. S. Lapp. in preparation). The results support the hypothesis that pl:C activates recipient NK cell activity. thereby mediating re.jcction of transplanted Bh donor cells. Furthermore. our observation that pl:c‘ induced protection is rcstrictcd to B6 (H-2Dh), and not seen when A strain (H-31Y) donors are used. is consistent with other ohscrvations of the HR phenomenon. Cantor (11 trl. (41) have previously shown that treatment of prospective donor mice with pl:C 3 day\ prior to utilization Icd to enhanced GVH reactivit). .4 few major differences exist between their studies and ours. Since thebe author\ used ;L parental-F, combination which does not display HR. they may be examining ;I different phenomenon from LI\. In addition. they used much higher do\e\ of pI:(‘ (2.5 to 5-fold higher) and newborn recipients. which are more sensitive to the GVH reaction than adult recipients. Furthermore. Cantor 01 trl. only mca~u~-ed splenomegaly. which WC have found to be an unreliable indicator of the GVH reaction (T. Ghayur-. T. A. Seemayer. and W. S. Lapp. manuscript irl preparetion). Other studies of treatment4 which alter NK activity during the GVH reaction have been reported. Treatment of GVH mice with IF (41) and ;tnti-;isi:do GM, (43). agents which augment and suppress NK cell activity. respectively. c;u~\es marked changes in the GVH reaction. In these studies (42. 331. treatment ~4 administered throughout the experiment, starting a few days prior to parental cell transfer and continuing until the animals were sacrificed. In the present study. pI:C was administered only once prior to GVH induction and this led to ;I clecrease in the severity of the reaction. The arnc pl:C treatment may have clifferent effects depending on the time of administration. It is possible that rcpetitive administrations of a single agent may superimpose more than one effect induced by that agent. For a more complete understanding of NK cell function during the GVH reaction a more defined use of modulating agents is required. ACKNOWLEDGMENTS We thank tance. and

Ailw Sandra

Lee Loy. Rosmarie James for excellent

Siegri4t-Juhn~tont. and wcretarial a\si\tancc.

I.eti

t’egorari

for careful

techmcal

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