T-Celll Recruitment Regulated by Prostaglandin-mediated System and its Role in Immune Response

Immunobiol., vol. 166, pp. 382-396 (1984) Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan

T-Cell Recruitment Regulated by Prostaglandin-mediated System and its Role in Immune Response Y. KOGA, K. TANIGUCHI, and K. NOMOTO Received December 20, 1983 . AcceptedJanuary 19. 1984

Abstract The dynamics of the number of T cells in spleen and the level of prostaglandin E2 in plasma were investigated serially in mice injected with Corynebacterium parvum. In the first few days, the level of plasma PGE2 increased but decreased to lower than the normal level thereafter. The absolute number of T ceUs in the spleen began to increase after the PGE 2 1evei dropped. But such an increase of T cells was not observed in ATx mice challenged with C. parvum. Moreover, replenishing the mice with exogenous PGEl in the period of low PGE2 halted selectively the increase of T cells in the spleen. This enlarged T cell subset responded to FHA, expressed Lyt~1 + 2 t-, and was sensitive to PGE 2 • And this T-cell subpopulation exerted a suppressive effect on antibody response in low PG environment, but lost its inhibitory effect in high PG milieu. These results suggested that an immature T cell subset is recruited from the thymus in a low PC state and participates as regulator cells in immune response at peripheral lymphoid organs.

Introduction The generation of immunocompetent T lymphocytes within the thymus involves complex processes that include the immigration of hematopoietic stem cells to the thymus, proliferation and functional changes of such cells, and emigration of the progeny of these cells to peripheral lymphoid tissues. Migration studies indicated that the majority of cells leaving the thymus home preferentially to the splenic white pulp and lymph node paracortex (1-2). Others also showed the migrating cells derived from the thymus cortex are still immature in phenotypes, and go mainly to the spleen (3). This cell population seems to constitute the pool of post-thymic precursor T cells in the spleen described by STUTMAN (4-5), however, the role of such a cell series in immune response in vivo remains unclear. The prostaglandins (PG) have been proposed as one of the intercellular humoral mediators that regulate immune responses (6 -7). Macrophages Abbreviations: PG = prostaglandin; INDO = indomethatin; SRBC = sheep erythrocyte; ConA = concanavalin A; PHA = phytohemagglutinin; LPS = lipopolysaccharide; C. parvum = Corynebacterium parvum; PFC = plaque-forming tell; ATx = adult thymec­ tomized; sham = sham-operated.

T Cell Recruitment and Prostaglandins· 383

produce PGs in response to a variety of exogenous stimuli including Corynebacterium parvum (c. parvum) (8), endotoxin (9), zymosan (10), and heterologous erythrocytes (11). It has also been reported that prolifera­ tion and differentiation of T cells are regulated by PG-mediated system (7). We already reported (12) that an administration of indomethacin (INDO), an inhibitor for PG-synthesis, decreased the level of PG in the plasma and increased the number of splenic T cells in nonnal mice but not in adult­

thymectomized (ATx) mice. The enlarged T-cell population consisted mainly of Lyt-1+2+ cells. These results suggested that the emigration of T cells from the thymus to spleen is affected by PGs. In this study, we administered killed-Co parvum, one of the potent immunomodulators, to mice and observed the level of PG in the plasma and the changes of T cells in the spleen. In this paper, we demonstrated that the T cell traffic from the thymus to spleen is regulated by PG-mediated system and plays an impor­ tant role in the immune response at peripheral lymphoid organs.

Materials and Methods Mice Males of inbred C3H/He mice were supplied from the Breeding Unit of Kyushu University and used for experiments at 8 weeks of age. Adult thymectomy (ATx) and sham operation (sham) were performed 7 days before the experiment. Cell idenrificauon Nucleated cells were obtained by squeezing the spleen and coumed with a hemocytometer. T and B lymphocytes were enumerated by the direct immunofluorescence method using FITC-conjugated monoclonal anti-Thy 1.2 antibody (Becton Dickinson, CA, USA) and FITC-labeled goat anti-mouse immunoglobulin serum (Cappel Laboratories, PA, USA) respectively. Spleen cells were also examined histologically by Giemsa or esterase staining for identification of polymorphonuclear leukocytes (PMN) and macrophages. Drugs and mieogens INDO or PGE 2 (Sigma Chemical Co., MO, USA) was dissolved in ethanol at 10 mg/ml and diluted with medium to desired concentrations before use. Concanavalin A (ConA, Sigma type IV), phytohemagglutinin (PHA, Sigma type III), and lipopolysaccharide (LPS, E. coli 01 I1; B4, Difco Lab. Mich., USA) were all stored at -20"'C and diluted to desired concentrations before use. A suspension of fonnalin-killed C. parvum (1M 1585) was supplied by Institute Merieux, Lyon , Prance. Stimulation of spleen cells with mieogen Cell suspension (5 x 10lt/ml) from the spleen was prepared in the medium which contained 5 % fetal calf serum,S X 10- 5 2-mcrcaptoethanol, 20 mM HEPES, 100 U/ml penicillin, and 100 f.1g1ml streptomycin (complete RPMI). The cell suspension (tOO~) was added to wells of a microplate (Falcon 3042, CA, USA) and an equal volume of medium containing mitogen was

384· Y. KOGA, K. TANIGUCHI, and K. NOMOTO added. PHA, ConA, and LPS were used at 5 ILg/ ml, 5 ILg/ml, and 10 Ilg/ ml, respectively, in the culture. The microplate was incubated for 48 hr in a humidified atmosphere of 5 % CO2 at 37°c' 20 III of medium containing 0.4 IlCi l H~thymidine was added to each well 6 hr before harvesting cells on filter papers. The samples were counted in a liquid scintillation counter as triplicate.

Production of PG in

VJ·tro

PGs were produced in a Falcon 3001 dish. Spleen cells (1 X 107) wcre cultured in a total volume of 2 ml of complete RPMI in an atmosphere of 5 % CO 2 at 37 °c' Two days later, at least two cultures per one group were pooled, centrifuged, and the 1 ml of supernatant was removed at once for the assay of PGE z•

In vitro generation of PFCs ro SRBC PFCs were generated in a Falcon 3002 dish. Spleen cells (2.5 X 107) and SRBCs (2.5 X 107) were cocultured in a total volume of 5 ml of complete RPMI in an atmosphere of 5 % CO 2 at 37°C. Five days later, at least two cultures per one group were pooled, washed, and the number of PFCs to SRBC per one culture was counted as triplicate by the method of CUNNINGHAM (13). Drugs were added at the beginning of cultures.

Treatment wirh antibody plus complement Anti~Thy 1.2 monoclonal antibody (Olac 1976 Ltd, England), anti~Lyt-1.1 monoclonal antibody, anti-Lyt-2.1 monoclonal antibody (Cedarlane Lab., Canada), and Low-Tox~M rabbit complement (Cedarlane) were used in this procedure. The detail was already described elsewhcre (12).

Enrichment of T cells Enrichment of T cells was performed using plastic surfaces coated with anti~mouse immunoglobulin antibody (14). Briefly, 2 ml of cell suspension (1 X 107 1ml in complete RPMI) was added to a culture flask (Falcon 3013) coated with F(ab'}2 fragment rabbit anti­ mouse IgG (heavy and light chains, Cappel Lab.). After incubation for 1 hr at room temperature, nonadherent cells were collected by rocking the flask and gende pipetting. The cell recovery was 20 to 30 %. Such cell population consisted of more than 70 % T cells and less than 5 '% B cells according to the immunofluorescence method.

Extraction and radioimmuno.assay (RIA) for

PG~

PGE2 was quantified using the method described by lnagawa (15). Briefly, blood specimens obtained from each group consisting of three mice were pooled and centrifuged at once to separate the plasma. 1 ml of the plasma was mixed with 20 ml chloroform: methanol = 2:1 , and shaked violently. After an elimination of crude sediment by a filter, these organic solvents were evaporated to dryness, dissolved in LP solution, and applied to Sephadex G-25 column soaked with UP solution. LP solution is the lower phase that is formed by mixing the foHowing solution, chloroform: methanol : HCI~acidified water (PH 2.0) = 200:100:75, by volume, and UP solution is the upper phase. The elute was dried, redissolved in carbon tetrachloride, and added to 10 % methanol-phosphate buffer. After shaking and centrifuga­ tion, the upper layer was collected, acidified with HCI, and then added to the ethyl acetate layer. After another shaking and centrifugation, the ethyl acetate layer was dried in vacuo. The residue was then applied to thin layer chromatography. The area corresponding to standard PGE 2 was scraped off, extracted with O.S % acetate-methanol, dried under the stream of N 2, and redissolved in assay buffer. Recovery of PGE 2 was assessed in separate samples by adding known 3H-PGE2 quantity before extraction procedures. Concentration of PGEz was then determined by RIA. Standard PGE2 and anti-PGEz antiserum was generously supplied by Ono Pharmaceutical Co., Osaka, Japan. Cross-reactivity of that antiserum to PGA, B F la and F2a was 8.4 % or less, and to PGE 1 was 53.3 %.

T Cell Recruitment and Prostaglandins . 385

Results

Plasma PGE2 and splenic cellularity in mlCe injected with C. parvum (Fig. 1)

Mice were injected intraperitoneally with 0.6 mg of C. parvum on day O. The level of plasma PGE, and the numbers of T and B cells were examined serially in these mice, thereafter. The PGE,-Ievel increased about twofold on day 1, however, decreased abruptly to less than half of the normal level on day 6. Such a declined state of PG in the plasma continued to day 20. The absolute number of T cells in a whole spleen exhibited no change until day 10, however, when it increased sharply from then on and reached to a plateau on day 20. It seemed to suggest that the preceding low level of PG in the plasma was concerned with the enlargement of T cells in the spleen. The

ng/ml • xl0- 7 00

15

10

5

o L--L____________L -__________-L____________ o

10

20

L-~--

30

day

Fig. 1. Profile of splenic T and B cells and plasma PG~. Donor mice were injected with 0.6 mg of killed C. parvum on Day o. The absolute number of T (0) and B (0) cells in the spleen. and the concentration of PGE2 (e ) in the plasma were examined serially on indicated days. Bars represent standard deviations (SD, n = 3).

Counted on day 20

± SD,

(+)

b per one spleen, Mean

~

n

(-) (+ )

Sham Sham ATx ATx

(-)

C. parvum challenge

Donor mlce

=

3

12.4

24.7 14.6 28.4

± 2.9 ± 2.0 ± 6.0

±

2.3 b

No. of nucleated cells (x 10')' 24 ± 2 24 ± 3 22 ± 2 14 ± 1

(%)

Table 1. Thymus-dependency of the increase of splenic T cell number

2.95 5.83 3.27 3.99

± 0.57 ± 0.77 ± 0.50 ± 0.74

(No.)

T cells

±4

± 5 34 ± 5 39 ± 2

41 32

(%)

5.07 7.99 5.05 11.13

(No.)

B cells

± 2.64

± 1.66 ± 1.12

± 0.52

4

4

±

N.D. 1

±1

2± 1

(%)

PMN

21

±4

8±2 15 ± 4

(%)

M0

~ ~

0

"S

0

Z

i"

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T Cell Recruitment and Prostaglandins' 387 Table 2. Change of plasma PGE 2 in ATx mice Days after C. parvum challenge

Plasma PGE, (ng/ml)

o

6.8 ± 1.6

1

14.2

7 20

8.1

± 2.7

3.6 ± 0.7

±

2.1

number of B cells increased from the start of C. parvum-challenge before the drop of PG-level in plasma.

Enumerations of T cells in ATx mice In a previous study (12), we have already observed the enlargement of splenic T cell population in mice whose level of PGE, in the plasma was decreased by an administration of INDO. But such an increase of T cells was not demonstrated in ATx mice. As shown in Table 1, an increase of the absolute number of T cells in the whole splenic cell population by challenge with C. parvum was detected in sham mice but not in ATx mice. Enlarge­ ment of the splenic T cell population by treatment with C. parvum was shown also to be thymus-dependent. After a treatment with C. parvum, the change of plasma PGE, levels showing an initial increase followed by a period of low levels was observed in ATx mice (Table 2) as well as in normal mice (data reported in Fig. 1). Thus, we concluded that the thymus was indispensable for enlargement of splenic T cell population in mice of which PG-milieu was low.

Replenishment with exogenous PCE, in mice at a low PC-level The next experiment was conducted in order to ascertain that the thymus-dependent increase of splenic T cells is induced in the low level of PGE, raised by a treatment with C. parvum. As exhibited in Fig. 1, the Table 3. Replenishment with PGE1 halts the increase of T cells

C. paJVum PGE2 challenge"

No. of nucleated treatmentb ceUs (x 10')'

T cells (%)

+ ++

+

11.5 n.5 17.5 8.5

± ± ± ±

2.2 J.2 4.8 0.7

24 17 20 26

± ± ± ±

3 1 2 3

B cells

(No.)

(%)

2.79 ± 0.87 5.76 ± 0.55

42 32 43 39

3.41

± 0.69

2.22 ± 0.15

± ± ± ±

(No.) 4 4 5 5

4.9 10.8 7.4 3.3

Challenged on day 0 200 J.&g of PGE1 were injected subcucaneously once a day from day 7 till day 16 , Counted on day 20 I

b

± ± ± ±

1.1 1.8 J.2 0.6

388 . Y. KOGA, K. TANIGUCHI, and K. NOMOTO

PGE,-level in the plasma of mice injected with C. parvum declined after day 6, so the mice were injected subcutaneously with 200 Itg of exogenous PGE, every day from day 7 to day 16. The number of splenic T cells of these mice was counted on day 20 (Table 3). The absolute number ofT cells was enlarged significantly on day 20 by injection with C. parvum (compare line 2 with I, P< 0.05). However, the replenishment with exogenous PGE, halted the enlargement of T cells in such C. parvum-treated mice (compare line 3 with 1, statistically insignificant). Exogenous PGE, itself exerted no effect on the number of splenic T cells in normal mice. The increase of B cell number was not stopped with exogenous PGE, (compare line 3 with I, P < 0.05).

Mitogen response and in vitro PGE, production by spleen cells from C. parvum-injected donors The effects of C. parvum on the mitogen response and production of PGE, in vitro by spleen cells were observed serially in normal mice injected with it on day a (Table 4). Mitogen responses to PHA, ConA, and LPS were inhibited markedly on day 7 and recovered almost fully on day 24. PGE,-production in vitro by spleen cells showed the same biphasic pattern of an initial increase on day 3, a decrease on day 7 as shown in the plasma of C. parvum-treated mice. When INDO (5 x 10- 6 M) was added to the culture at first, the PGE, generated was less than 1 ng/ml in any of these cultures (data not shown).

Effect of A Txon mitogen responses and their sensitivities to PGE, (Table 5) We already demonstrated in Table 1 that an enlargement of T cell population in the spleen was not observed in ATx mice challenged with C. parvum. The responses to PHA and ConA on day 7 were depressed markedly both in sham and ATx mice challenged with C. parvum on day a Table 4. Time courses of mitogen response and in vitro PGE2 production Days after C. parvum challenge

PHN

0 3 7 24

91.7 ± 15.5' 28.8 ± 1.8 4.3 ± 0.6 86.4 ± 3.2

ConAb

PGE,

LPS'

DPM (X W') 193.9 143.4 56.9 188.5

± ± ± ±

15.3 4.8 5.1 3.7

(ng/ml) 96.7 83.8 18.3 88.5

± ± ± ±

2.5 2.0 3.2 7.0

4.8 9.8 2.5 6.2

± ± ± ±

0.9 2.9 0.6 2.1

Note: Splecn cells were removed from C. parvum-challenged donor mice at indicated intervals and cultured in vitro for mitogen response or PGE1 production .

•5 b

~g/ml

5 ~g/ml

, 10 ~g/ml d Mean ± SD, n

=

3

(- )

Sham Sham

(+) (+) (+) (+)

Exp. 2, (Day 7) Sham ATx Sham ATx

±

2.2

± lOA

ConA ConA

3.7 6.0 52.6 99.8

± ± ± ± 0.2 1.3 4.0 4.2

5.8 7,8 58.2 105.5

± 0.4 ± 1.4 ± 3.7 ± 12.3

95.6 ± 6.9 91.7 ± 4.9 96.5 ± 4.0 79.8 ± 4.3

89.9 ± 11.8 89.5 ± 4A 96.0 ± 10.7 80.7 ± 3.2

LPS LPS LPS LPS

PHA PHA

185 .1 ± 11.4 168.8 ± 9.9 176.5 ± 5.6 147.0 ± 10.0

72.1 ± 3.2 58.9 ± 1.0 78.3 ± 2.4 20.5 ± 2.3

INDOh

± 9_6

± 0.8

± 3.7 ± 004

N.D. N.D. N.D. N.D.

N.D. N.D. N.D. N.D.

173 .4 ± 6.7 183.1 ± 7.9 174.6 ± 4.1 137.9 ± 3_5

39.2 28.4 57.8 14.6

DPM (x W')

INDO + PGE,

5 x 10-' M

added to the culture

170.8 ± 1.1 183.1 ± 9.6 149.3 ± 1.3

± 5.2 ± 4.5 180.0 ± 7.0

59.9 56.7 65.5 20.5

(-)

Drugs~

ConA ConA ConA ConA

PHA PHA PHA PHA

Mitogen

2.1 3.6 63_5 79.1

87.6 85.2 108.1 78.3

171.9 166.6 180.0 137.0

27.1 18.6 42.7 10.6

5.2 ± 0.9

±

4_8

± 0.2 ± 0.4

±

± 5.7 ± 4.1 ± 9.6

± 11.2

4.4

± 10.2 ± 9.6 ± 6.7

±

+ PGE,

± 1.6 ± 3.6 ± 004

INDO

5 X 10". .7 M

Note: Donor mice with adult-thymectOmy or sham operation on day -7 were challenged with C. parvum on day 0, and their spleens were removed for mitogen response on day 7 (Exp. 2) or day 20 (Exp. 1). a Added at the beginning of culture. b 5 x 10-4> M.

(+ )

ATx ATx

(-)

(+)

(+)

(- )

ATx ATx

(+)

(- )

(-) (+) (- ) (+ )

C. parvum challenge

Sham Sham

Sham ATx ATx

Sham

Exp. I, (Day 20)

Donor mice

Table 5. Depressed response to PHA in ATx mice and its sensitivity to PGE

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390 . Y. KOGA, K . T ANIGuclII, and K. N OMOTO

Table 6. Effect of T cell enrichment on mitogen response Donor mice

C. parvum challenge

Mitogen response

PHA Sham

Sham

ATx ATx

+ +

145.2 165.0 147.6 109.2

ConA (DPM X 10-')

± ± ± ±

12.8 3.2 8.7 8.6

194. 1 189.9 172.7 180.7

± ± ± ±

7.9 11.1 10.9 3.2

Note: Spleen cells from donor mice with or without C. parvum challenge were removed on day 20 and fractionated for enrichment of T cells before culture.

(Table 5, Exp. 2). PHA response in ATx mice was still depressed on day 20, although the response in sham mice recovered fully on day 20 by challenge with C. parvum (Table 5, Exp. I, compare lines 1 and 3 with lines 2 and 4, respectively). On the other hand, the responses to ConA in sham and ATx mice recovered to the level by untreated spleen cells on day 20. LPS response was not modified by C. parvum-challenge 20 days before or to ATx. Addition of INDO to cultures did not modify s ignificantly the responses to PHA, ConA, and LPS by spleen cells of any donors. On the other hand, the addition of PGE, at the physiological concentration (5 X 10- 7 M) to the cultures suppressed PHA response by spleen cells of sham mice challenged with C. parvum 20 days before or nontreated sham mice (Table 5, Exp. I, lines 1 and 2), but not ConA and LPS responses by such cells (Table 5, Exp. 1, lines 5, 6 and 9,10). ATx modified scarcely the effect of PGE, added on ConA or LPS responses. The T cell-enriched population of ATx mice challenged with C. parvum 20 days before showed a decreased PHA response as compared with the response by those of C. parvum-treated sham mice (Table 6). Both enriched T cell populations from A Tx and sham mice equally consisted of 70-75 % T cells. ConA response was not modified by C. parvum treatment also in this experiment with T cell-enriched population. The results in Tables 5 and 6 suggested that a depressed PHA response in C. parvum-treated mice may be due to a decrease of a T cell subset which responds to PHA and is sensitive to PGE, .

Treatment of responder cells with anti-Lyt antibody plus complement Lyt-phenotypes of responder cells to mitogens were analyzed by treat­ ment with anti-Lyt antibody plus C' (Table 7). Spleen cells of donor mice challenged with C. parvum 20 days before or not were treated at the beginning of culture. The proliferative response to PHA by the spleen cells from C. parvum-challenged mice was abrogated profoundly by treatment with anti-Lyt-I plus C' and also with anti-Lyt-2 plus C' likewise. On the

65.8 ± 3.5 187.9 ± 13.8 57.6 ± 6.7 177.3 ± 17.8

65.2 ± 4.1 166.1 ± 37.2 93.1 ± 7.8 180.2 ± 22.7

PHA ConA PHA ConA

C. parvum C. parvum Nil Nil

6.8 ± 0.7 18.1 ± 1.9 18.1 ± 1.2 63.5 ± 7.0

(DPM x 10-' ) 22.4 156.9 28.7 133.5

± ± ± ±

0.9 23.3 1.3 14.3

Spleen cells treated with aLyt-1+C' aLyt-2+C'

4.0 21.6 4.3 24.3

± 0.4

± 1.9 ± 0.2 ± 2.7

aThy-1+e'

Note: Donor mice were inj ected with C. parvum 20 days before. Spleen cells were treated with antibody plus C' before culture. Cell number was adjusted before treatment and not readjusted after treatment.

C'

Nil

Mitogen

Donor mice treated with

Table 7. PHA responsive cells were Lyt-l +r

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392 . Y. KOGA, K. TANIGUCHI, and K. NOMOTO

other hand, the response to Con A by such cells was eliminated with anti­

Lyt-l plus C but not with anti-Lyt-Z plus C. Thus, it was indicated that mast of the PHA-responsive cell population in spleens from C. parvum­ injected mice express both Lyt-l and Lyt-Z markers. However, the ConA­ responsive cell population bears only Lyt-1.

In vitro antibody response to SRBC

It was demonstrated by now that one T cell subset, which expresses Lyt1+Z+, responds to PHA and is sensitive to PGE, for its proliferation, appears in the spleen of mice about ZO days after challenge with C. parvum. But such a T cell subpopulation was detected little in the spleen of ATx mice after C. parvum-injection. So the experiment shown in Table 8 was

conducted to elucidate the role of such T cells on immune response. The dose of exogenous PGE, added (5 X 10- 7 M) in this culture for antibody response, under the blockade of synthesis of endogenous PGs by INDO, had inhibited only the proliferative response to PHA but not that to ConA or LPS by spleen cells as shown in Table 5. Thus, it was expected that only the T cell subset, which responds to PHA and bears the Lyt-l +z+ phenotype, is selectively inhibited to proliferate in this culture by the addition of such a dose of PG E. When C. parvum was given ZO days before, antibody response in vitro to SRBC was suppressed Table 8. Different effects of PGEl added on antibody response in vitro Donor mice

C. parvum challenge

Drugs' added to the culture

INDO b

PGE{ (-)

PFCs/culture (x 10')

% increased

(%)

Sham Sham Sham

(+) (+) (+)

(-)

(+) (+)

(-)

59.6 ± 5.3 39.8 ± 0.8 51.1 ± 1.6

22

ATx ATx ATx

(+) (+ ) (+)

(-)

(-) (-)

70.0 ± 4.5 39.4 ± 4.3 39.4 ± 6.4

0

Sham

(-) (-) (-)

(-) (-)

56.3 ± 1.4 69.0 ± 3.7 44.5 ± 5.7

-35

49 .3 ± 4.2 101,4 ± 8.5 69.9 ± 9.8

-31

Sham Sham

ATx ATx ATx

(- ) (-) (-)

(+) (+)

(+)

(+)

(+) (+)

(-)

(-)

(-)

(+) (+ )

(+)

(+)

(-)

Note: Spleen cells were removed on day 20 from donor mice with or without C. parvum challenge and cultured for 5 days to generate PFCs to SRBC. Added at the beginning of culture. b 5 x 10~ M. 1

c

5 X 10-7 M. % increase = (No. of PFCs in the culture with INDO and PGE 2

- No. of PFCs in the culrure with INDO only)/No. of PFCs in the culture with INDO and PGE2 ·

d

T Cell Recruitment and Prostaglandins . 393

by blockade of endogenous PGE, synthesis with INDO both in sham and ATx mice (compare lines 1 and 4 witb lines 2 and 5, respectively). Addition of exogenous PGE, to such cultures restored the response by spleen cells of C. parvum-treated sham mice but not the response by those of C. parvum­ treated ATx mice (compare lines 2 and 5 with lines 3 and 6, respectively). When C. parvum was not given, addition of INDO to cultures augmented antibody response by spleen cells of both sham and ATx mice (compare lines 7 and 10 with 8 and 11, respectively). The addition of exogenous PGE, to such cultures, however, suppressed such an augmented response (com­

pare lines 8 and 11 with lines 9 and 12, respectively). When spleen cells of C. parvum-treated ATx mice were examined for their responsiveness to PHA,

the addition of INDO to the culture did not alter further as shown in Table 5. However, antibody response by such cells was suppressed by the addition of INDO to the culture as shown in Table 8. Such a discrepancy between PHA and antibody response may be ascribed to that the antibody response requires also the cells other than the cells required for PHA response in ATx mice.

Next, antibody response was examined by the culture composed of enriched T cells (5 X 10') from such donor mice, and B cells prepared by treatment of normal spleen cells with anti-Thy-l plus C' (2 X 10'), and 5RBC (2.5 X 10') (Table 9). The generation of PFCs was augmented significantly by PGE, in the culture consisting of T cells from C. parvum­ treated sham mice, but not in the culturc composed of T cells from C. parvum treated ATx mice. 50 it was indicated that a PG-sensitive, Lyt1+2+, T-cell subpopulation, which may be recruited from the thymus, exerted a suppressive effect on antibody response in a low PG environment

raised by the addition of INDO. In the higher PG-milieu (5 X 10-' M) raised by exogenous PGE" however, this T-cell subset could not act as regulator cells, and exerted no suppressive effect on T-cell-dependent antibody response. Table 9. T cells exert different effects Donor mice"

Drugs added to the culture INDO'

(- )

PFCsl cuhure (x 10' )

Sham Sham Sham

(- )

(+ )

(-)

(+)

24.3 13.8 23.5

± 1.7 ± 2.0 ± 3.2

ATx ATx ATx

(-) (+)

(-)

21.1

± 2.6

11.1 13.4

± 2.1

(+) (+)

(- ) (+)

• Challenged with C. pa.rvum 20 days before. 5 X 10..{, M. 7 c 5 X 10- M. b

±

% increase

71

1.2

20

394 . Y. KOGA, K. TANIGUCHI, and K. NOMOTO

Discussion

The dynamics of change in number of T cells in the spleen and the level of PGE, in the plasma was investigated serially in mice injected with C. parvum. The level of plasma PGE, was increased in the first few days but decreased abruptly to lower than the normal level on day 6 and maintained at such lower levels for 2 weeks thereafter. The absolute number of T cells in the spleen began to increase on day 10, namely, several days after the drop of PGE,level, and reached a plateau on day 20. The number of B cells increased from the beginning before a decline of PGE, . In ATx mice, such an increase of T cells was not induced by injection with C. parvum, although the same pattern of plasma PGE,-dynamics was observed. Moreover, replenishing the mice with exogenous PGE, in a period of low PGE, halted selectively the increase of T cells in their spleens. In a previous paper, we already reported that aT-cell population consisting mainly of Lyt-l +2+ cells in the murine spleen was increased by administration of INDO, a specific inhibitor for PG synthesis which decreased the level of plasma PGE, significantly. In ATx mice, an enlargement of T-cell popula­ tion was not observed after administration of INDO. So we suggested that the increased T cells in the spleen were recruited from the thymus by a PG­ mediated mechanism. In this study, we could also demonstrate both thymus dependency and participation of low PG level on an increase of splenic T cell population in mice challenged with C. parvum. The enlarged T cell population in this experiment seemed also to consist of Lyt-l +2+ cells. Taken together, this finding firmly indicated that the thymus-depen­ dent increase of splenic T-cell population depends upon the PG-mediated system. Others (16-17) reported that activated macrophages from mice inoculated with C. parvum before 7 days exhibited a markedly decreased release of PG in vitro than did resident macrophages. And they concluded that a diminished capacity for the synthesis of arachidonic oxygenation products is a general property of various elicited populations of mouse macrophages when compared with resident macrophages. In our study, the amount ofPGE, produced in vitro (Table 4) was correlated with the level of PGE, in the plasma from the same donor mice. So the drop of PG-level in the plasma from mice inoculated with C. parvum was considered to reflect the stage of a decreased release of PG by those activated macrophages. Proliferative responses of splenic cell population from C. parvum­ injected mice to mitogens including PHA, ConA, or LP5 were diminished markedly on day 7 and recovered well on day 20. But in ATx mice only, the response to PHA did not recover fully on that day. Moreover, such a PHA­ responsive T-cell sub population was blocked to proliferate at the phy­ siological concentration of PGE, in vitro. Thus, it was concluded that in low PG-milieu, a T cell subpopulation, which may be recruited from the thymus, was increased in the spleen of thymus-intact mice. This T cell subset expresses Lyt-l +2+ phenotype, responds to stimulation with PHA,

T Cell Recruitment and Prostaglandins· 395

and is sensitive to PGE, for its proliferation. STUTMAN (4-5) proposed aT cell subpopulation called «post-thymic precursor T cells» in the spleen, the cells of which are considered to have emigrated from the thymus, spleen­ seeking, and bear immature characters such as Lyt-l +2+ phenotype and to be cortisone-sensitive. These immature cells are immunologically incompe­ tent at firS!; however, they gain the ability to respond to PHA later after the differentiation in the peripheral lymphoid organs. The PG-sensitive Lyt1+2 + cells observed in our system may be those immature cells that emigrated newly from the thymus to the spleen after the splenic T cells, which resided there earlier, exhausted their immunological reactivity to further stimulation, s uch as lectin, by first an overwhelming stimulation with C. parvum. The signal of such a T cell recruitment may be given by macrophages which produced little PG and reflected its exhaustion by a low PG level in the plasma. With the level of PGE, expected to block selectively the activity of such PG-sensitive cells, antibody response to SRBC was augmented in C. parvum-treated sham mice but not in C. parvum-challenged ATx mice, which do nOt contain those regulator cells. CEUPPENS et al. (18) also reported the enhancing effect of a low dose of PGE, (3 X 10- 8 M) which inhibits suppressor T cells in antibody response in vitro. In our system, these regulator T cells exerted a suppressive effect on antibody response to T-dependent antigen SRBC in low PG-envitonment, however, lost such an inhibitory effect in high PG-milieu. This will be realized by an augmented production of PGE, by newly arriving macrophages at the site of further inflammation or antigen stimulation in vivo. References I. GOLDSCHNEIDER, I., and D. D . MCGREGOR. 1968. Migration of lymphocytes and lhym()(;yt~s in the rat. I. The route of migration from blood to spleen and lymph nodes. J. Exp. Med. 127: 155. 2. PARROTT, D. M. V., and M . A. B. DESOUSA. 1971. Thymus-dependent and thy mus­ independent populations. Origin, migration patterns, and life span. Clin . Exp. ImmunoL

8: 663. J. D UR KIN H . G .• 1- A. CARBONI. and B. H . W AKSMAN. 1978. Antigen-induced increase in migration of large conical thymocytes (regulatory cells?) to the marginal zone and red pulp of the spleen. 1- Tmmunol. 121: 1075. 4. STUTMAN, O . 1977. T wo main features of T-cell development: Th ymus traffic and postthymic matur2tion. Contemp. Top . Immunobiol. 7: I. 5. STIITMAN, O. 1978. Intrathymic and extrathymic T cell maturation . Tmmunol. Rev. 42: 138. 6. SENSON, W. E, and C. W. PARKER. 1980. Opinio n, prostaglandins, macrophages, and immunity . J. Immunol. 125: 1. 7. GOODWIN,]. G., and o. W. WEBn. 1980. Regulation of the imm une response by prostaglandins. C lin . Immunol. lmmunopathol. 15: 106. 8. GRIMM, W. , M . SEITZ, H . KIRCHNER, and D . GEMSA. 1978. Prostaglandin synthesis in splcen cell cultures o f mice injected with Corynebacterium parvum. Cell. Immunol. 40: 419 .

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9. KURLAND, J. 1., and R. BOCKMAN. 1978. Prostaglandin E production by human blood rnonocytes and mouse peritoneal macrophages. J. Exp. Med. 147: 952. 10. HUMES, J. L., R. J. BONNF.Y, L. PEtUS, M. E. DAHLGREN, S. J. SADOWSKI, f. A. KUEHl., Jr. and P. DAVIES. 1977. Macrophages synthesize and release prostaglandins in response to inflammatory stimuli. Nature 269: 149. 11. WEBB, D. R .• and P. L. OSHEROFF. 1976. Antigen stimulation of prostaglandin synthesis and control of immune responses. Proe. Nat. Acad. Sci. 73: 1300. t2. KOGA, Y., T. KAZurO, C. Kuno, and K. NOMOTO. 1983. Thymus-dependent increases in splenic T-cell population by indomethacin. Cell. lmmunol. 75: 43. 13. CUNNIGHAM, A. J., and A. SZENBERG. 1968. Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14: 599. 14. GEORGE. K. L., and R. KAMIN. 1980. Cell separation. Separation of T and B cells using plastic surfaces coated with anti-immunoglobulin antibodies. In: Selected Methods in Cellular Immunology. B. B. MlSHELL and S. M. SHIIGI, eds. W. H. FREEMAN and Company. p. 227-234. 15. INAGAWA, T. 1982. Assay of prostaglandins. Journal of Medical Technology Gapanese) 26: 135_ 16. HUMES, J. L., S. BURGER, M. GALAVAGE. F. A. KUEHL, Jr., P. D. WIGHTMAN, M. E. DAHLGREN, P. DAVIES, and R. J. BONNEY. 1980. The diminished production of arachidonic acid oxygenation products by elicited mouse peritoneal macrophages: pos­ sible mechanisms. J. Immuno!. 124: 2110. 17. SCOIT, W. A., N. A. PAWLOWSKI, H. W. MURRAY. M. ANDREACH, J. ZRTKF., and Z. A. COHN. 1982. Regulation of arachidonic acid metabolism by macrophage activation. J. Exp_ M,d_ 155: 1148_ 18. CEUPI'ENS, J. L., and j. S. GOODWIN. 1982. Endogenous prostaglandin E z enhances polyclonal immunoglobulin production by tonically inhibiting suppressor T cell activity. Cell. Immunol. 70: 41. Dr. Y. KOGA, Department of Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1, Maidashi, Higashi-ku, J-Fukuoka 812, Japan