Clin. exp. Immunol. (1990) 82, 445-449
Effects of FK506 on rat thymus: time-course analysis by immunoperoxidase technique and flow cytofluorometry K. TAKAI, K. JOJIMA, J. SAKATOKU & T. FUKUMOTO* Department of Urology and * First Department of Anatomy, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan
(Acceptedfor publication 4 July 1990)
SUMMARY The effect of administration of FK506 at 1 mg/kg body weight for 14 days on rat lymphoid tissues, especially the thymus, and recovery after discontinuation of treatment, were investigated by the immunoperoxidase technique and flow cytofluorometry using monoclonal antibodies OX6, OX7, OX8, OX 18 and W3/25, reactive with rat lymphocytes. Marked reduction of the thymic medulla upon treatment was clearly demonstrated by staining with OX 18 and OX6. Changes produced by FK506 were also observed in the cortical area of the thymus, and were especially marked in the subcapsular area and around blood vessels. Eventually, the thymic cortex appeared patchy, this change being maximal 14 days after the start of administration. Obvious restitution of the thymic medulla was evident about 14 days after withdrawal of FK506. Flow cytometric analysis of the thymus showed that the percentages of cells labelled positively with OX7, OX8 and W3/25 were increased with FK506 treatment, and recovered to the normal levels soon after withdrawal. Furthermore, the peak of fluorescence intensity of OX7+, OX8+ and W3/25+ cells showed a temporary shift to the right during FK506 treatment; however, the peak of fluorescence intensity of OX 18 + cells showed a temporary shift to the left. Treatment with FK506 also produced a significant change in 3H-thymidine uptake by thymocyte. These results suggest that FK506 may inhibit the proliferation, maturation and differentiation of thymocytes. However, thymocytes prepared from FK506-treated rats and labelled with FITC behaved similarly to rat thymocytes in normal recipient rats. This suggests that during FK506 treatment thymocytes may retain their potential for peripheral mobilization.
Keywords FK506 thymus monoclonal antibodies immunohistochemistry flow cytofluorometry
INTRODUCTION FK506 (formerly designated FR900506) was isolated from Streptomyces tukubaensis strain 9993 in the laboratories of Fujisawa Pharmaceutical Co. (Osaka, Japan) in 1984. It has a macrolide chemical structure with a molecular weight of 822. In vitro experiments have demonstrated that the agent inhibits interleukin-2 (IL-2) production and generation of cytotoxic T cells at a concentration 100 times lower than cyclosporin A (CyA) (Sawada et al., 1987). In vivo studies on the effects of FK506 in organ transplantation have demonstrated a remarkable immunosuppressive potency and prolongation of allograft survival time in rats with heart and renal transplants (Ochiai et al., 1987; Collier et al., 1987). In relation to the toxicological and histopathological effects of FK506, one study has shown lymphocytic depletion in Correspondence: K. Takai, Department of Urology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan.
445
the rat thymic cortex (Nalesnik et al., 1987). According to subsequent studies, however, a striking reduction in the size of the thymic medulla with increased macrophage phagocytic activity in the cortex has been observed after FK506 treatment in rats (Stephen et al., 1989). T cells develop in the thymus and may mature as they migrate from the cortex to the medulla and then to the periphery, acquiring OX18 antigen and selectively losing and acquiring W3/25 and OX8 antigens. Monoclonal antibodies (MoAbs) against these surface antigens have been useful for distinguishing thymocyte subpopulations in rats (Mason et al., 1983). We have recently analysed the effect of CyA on the rat thymus, using the immunoperoxidase technique and flow cytofluorometry (Tanaka et al., 1988). Here, using a similar technique, we investigated in more detail the effect of FK506 on the thymus and its recovery after withdrawal of FK506, using MoAbs. We also analysed the effect of FK506 on spleen in relation to effect in the thymus, using flow cytofluorometric
K. Takai et al.
446
analysis of thymus. We present the results here and discuss the effect of FK506 on rat thymus tissue in relation to its
450
immunosuppressive activity.
400 -
w
E
MATERIALS AND METHODS Animals For analysis of the thymus and spleen, inbred male LEW (RT-1 ) rats, aged 10 weeks (mean body weight 275 g) were used. They were obtained from Seiwa Experimental Animals, Fukuoka, Japan, and were kept in our animal colony under routine laboratory conditions with free access to food and water.
c52 350 a)
E 300
2 _H250
200 L
FK506 administration The first day of administration was taken as day 0 of the experiment. LEW rats received FK506 (1 mg/kg body weight) by daily intramuscular injection for 14 days (days 0 to 13) into one of the four limbs, with daily rotation.
Experimental protocol On days 0, 3, 7, 10, 14, 21 and 28 the rats were killed by ether anaesthesia and tissues were removed for immunohistochemistry (thymus and spleen) and flow cytofluorometry (thymus). Antibodies MoAbs used for identifying rat lymphocyte surface antigens were W3/25 (CD4), T helper cells (Williams, Galfre & Milstein, 1977) and macrophages (Jefferies, Green & Williams, 1985); OX8 (CD8), T cytotoxic/suppressor cells (Brideau et al., 1980); OX18, class I MHC gene product (Fukumoto, McMaster & Williams, 1982); OX6, class II MHC gene product (McMaster & Williams, 1979); OX7, Thy-1.1 antigen (Mason & Williams, 1980). These specific MoAbs (from ascites fluid) were purchased from Sera Labs (Sussex, UK). FITC-conjugated sheep F(ab')2 anti-mouse IgG was also obtained from Sera Labs. Indirect immunohistochemical staining FK506-treated rats were killed under anaesthesia, and the thymus was frozen. Immunohistochemical staining of the thymus and spleen was performed as described previously (Fukumoto et al., 1982). Briefly, frozen sections of the thymus and spleen were reacted with each MoAb and further stained by the ABC method using a mouse IgG Vectastain ABC Kit (Vector Labs, Burlingame, CA), using the MoAb at 1/10 dilution in phosphate-buffered saline (PBS). Flow cytofluorometry Single-cell suspensions were obtained by pressing the thymus through a stainless steel mesh into RPMI 1640 (Flow Labs, North Ryde, Australia) supplemented with 5% heat-inactivated fetal bovine serum (Flow Labs). Erythrocytes were lysed with hypotonic ammonium chloride. Cells from the thymus were labelled with MoAb and FITC-conjugated secondary antibody, and analysed by flow cytofluorometry (EPICS-753; Coulter Electronic Laboratories, Hialea, FL) as described by Fukumoto et al., (1982). The labelled cells were then analysed. To clarify the fate of FITC-labelled thymocytes in normal recipient rats, single-cell suspensions of thymus were obtained as described above. Thymocytes were labelled with FITC using the method described by Butcher & Weissman (1980), then 1-5 x 108 FITC-labelled thymus cells from FK506-treated and
0
3
7
14 Time (days)
21
28
Fig. 1. Thymus weights of FK506-treated rats (m) or control rats (0) at various times during the experiment. The mean value of four thymus weights (±s.e.m.) is shown in each case. Control and FK506-treated groups were compared by Student's t-test. The decreases in mean thymus weight on days 14 and 28 are statistically significant: *PN-
1
thymus sections
of frozen
Immunoperoxidase staining patterns
from FK 506-treated rats labelled with
strongly
Note that
!
observed in the medulla (M) in controls.
with OX 1 8
0X6
or
were
Day O
(b),
4>
-.
Day 3
observed in the cortex
Day 7
a)
(C) in controls (a and c). Marked reduction of the thymic medulla (M)
C
were
the the
specific staining
observed with
thymic
OX 18
with
medulla and reduction of 0X6
thymic
remarkable
cortex
appeared patchy (d,
in
subcapsular
the
(e and f). Magnifications: a-d,
medulla had almost cortex was
19;
e
and
area
x
+
x
e,
day
on
area are
f).
and
around
47; f,
the medulla continued until
day
14
This
100
positivity for
treatment
evident, eventually The
reduction
blood
vessels
resulted in
| .-,'~I|.
is
(Fig. 2d).
On
patchy reduction
day 21,
of
the
-.---'-
..-;,
--
r; cot-,
.
Day 21 Day 28
,,
reduction of
thymic
0X6.
Day 14
,a)
(V)
|!
complete
OX18 and
J0 a)
190.
x
after cessation of treatment, the reconstituted
FK506
Atrophy of
completely atrophied by day 7, whereas
comparatively preserved.
showed normal
3 (b).
7
days
Fluorescence intensity
medulla
Additionally,
OX6+
area
in
Fig. 5. Fluorescence profiles of thymocytes labelled with OX8 in FK506treated rat thymus.
the cortex (Fig. 2d, 2e, 2f). The reduction was especially remarkable in the subcapsular area and around blood vessels (Fig. 2e, 2f).
[
0
-T
90 [
Flow-cytofluorometric analysis of thymus By scatter analysis, thymocyte size was invariable among the samples from day 0 to day 28. The percentages of cells positively labelled with OX7, OX8 and W3/25 increased with FK506 treatment, and recovered to
4-
a1)
a)
80p 0
the normal levels 70
[
87-3 +0-8, 91-4 + 2-9, 96-1 +0 7, 74-5 + 3-0 and 78-4+ 8-3, respectively, compared with 75-7 + 5-3 for controls (day 0). The percentages (±s.d.) of OX8+ cells on days 3, 7, 14, 21 and 28 were 79 9 + 2-5, 89 9 + 1 9, 94-6 + 2-2, 83-8 + 2-1 and 75-6 + 5-0, respectively, compared with 72-4 + 4 9 for controls (day 0). The percentages (±s.d.) of W3/25+ cells on days 3, 7, 14, 21 and 28 were 78-2 + 1-5, 87-5 +0-8, 91-7 +±40, 73-2 + 5 2 and 69 8 +0 6, respectively, compared with 74-6 + 3 -7 for controls (day 0). were
0
3
7
14
21
28
Time (day)
Fig. 3. Immunophenotypic analysis of rat thymus. 1rhe percentages of positive cells labelled with OX7 (-), OX8 (0) and W'3/25 (0) increased with FK506 treatment, and recovered to the normail levels soon after FK506 withdrawal. The value for each normal rat tl hymus around this age were almost similar to those of day 0.
after drug withdrawal (Fig. 3). The OX7+ cells on days 3, 7, 14, 21 and 28
soon
percentages (±s.d.) of
448
K. Takai et al. re 'ah .
I\
P,. ,,,^ v Day 21
Day 28 Fluorescence intensity
Fig. 7. Fluorescence profiles of thymocytes labelled with OX18 in FK506-treated rat thymus.
The fluorescence profile of thymocytes from normal adult a wide range of fluorescence intensity. The peak of the fluorescence intensity was shifted to the right with FK506 treatment on day 3. The maximal shift was observed on day 14. However, after cessation of FK506, the peak shifted back to coincide with the peak of the control until day 28 (Fig. 4). Analysis of OX8 + thymocytes from normal adult rat showed that they constituted a major population. The peak of the fluorescence intensity was shifted to the right with FK506 treatment, and after withdrawal the peak shifted back to coincide with that of the control (Fig. 5). The fluorescence profiles of W3/25 in normal adult rats showed that most thymocytes were W3/25 +. The peak of the fluorescence intensity was shifted to the right with FK506 treatment, and after cessation the peak shifted back to coincide with that of the control (Fig. 6). About 40% of the thymocytes of normal adult rats were OX 18+ and showed a wide range of fluorescence intensity. The rats, stained with OX7, showed
percentage of positive cells labelled with OX 18 appeared not to change. However, FK506 treatment resulted in a decrease in the proportion of the brightly OX 18 positive cells (Fig. 7).
FITC-labelled thymocytes The percentages (± s.d.) of FITC-labelled thymocytes from FK506-treated rats in normal recipient spleen, thymus and cervical lymph nodes were 6-1 + 1-5, 0 7+0-1 and 1 4+0 2, respectively. The percentages of labelled thymocytes from control rats in normal recipient spleen, thymus and cervical lymph nodes were 5-1 + 1 0, 0 8 + 0 1 and 2-2 + 0 5, respectively. The localizations of the FITC-labelled thymocytes from FK506-treated and normal rats were not significantly different. Immunohistochemical analysis of spleen FK506 treatment resulted in slight reduction in number of OX8+ or W3/25+ cells in periarterial lymphocyte sheath of spleen. 3H-thymidine uptake FK506-treated thymocytes showed low 3H-thymidine uptake compared with controls (Fig. 8). DISCUSSION
The thymus is an important organ for immune responses, especially cellular immunity. Thymocytes proliferate and mature in the thymus. Cortical thymocytes are generally considered to be less mature and less differentiated than medullary thymocytes (Mason et al., 1983). Here we investigated the effect of FK506 on thymocytes after intramuscular injection of the drug. Treatment with FK506 produced the increases in the percentages of OX7 +, OX8 + and W3/25 + cells (Fig. 3). This was followed by rapid recovery after cessation of treatment. This
449
Effects of FK506 on rat thymus increase of OX8+ and W3/25+ thymocytes in the thymus suggests that double-positive thymocytes may increase in the thymus during FK506 treatment. A relative increase of brightly fluorescent OX7 + cells during FK506 treatment was observed by flow cytofluorometry (Fig. 4). This increase may suggest an increase of Thy-i antigen per thymocyte, since the size of the thymocytes during FK506 treatment did not change remarkably. Accordingly, this may in turn suggest an increase of immature thymocytes (Williams, 1982). On maturation, the expression of MHC class I antigen on thymocytes is increased in the thymus (Fukumoto et al., 1982). We observed a relative decrease of bright OX 18 + cells during FK506 treatment (Fig. 7), perhaps suggesting a decrease of class I antigens per thymocyte. Treatment with FK506 may interfere with cell maturation in the thymus, which would support the above conclusion, since the amount of MHC class I antigen per cell is thought to increase in parallel with thymocyte maturation (Fukumoto et al., 1982). On maturation, the surface antigens of thymocyte are considered to change from CD4-CD8- (double negative) to CD4+CD8+ (double positive) in the thymus, and then to change from CD4+CD8+ to CD4+CD8- or CD4-CD8+ (single positive) (Mason et al., 1983). Thus, it is possible to consider the double-positive thymocytes are the immature thymocytes. More than 80% of thymocytes of normal rats are usually double-positive immature types (Fukumoto et al., 1982). In the FK506-treated thymus (as shown in Fig. 3 and discussed) double-positive immature population were also the main population of the thymocytes. When these FK506-treated thymocytes, which are mostly double-positive types, were analysed by EPICS, we observed a relative increase of brightly fluorescent W3/25 + and OX8 + cells (Figs 5 and 6). These mean an increase of CD4 and CD8 antigen per double-positive thymocytes. These facts may suggest that in FK506-treated thymus there exist more immature thymocytes than in normal thymus, and FK506 interferes with thymocyte maturation. Treatment with FK506 resulted in marked reduction of the thymic medulla, which was examined by specific staining with OX18 and OX6 (Fig. 2b, 2d). Additionally, FK506 treatment resulted in marked reduction of the OX6+ area in the cortex (Fig. 2e, 2f). The reduction was especially remarkable in the subcapsular area and around blood vessels, and eventually the thymic cortex appeared patchy. Thus, the OX6- area increased during FK506 treatment. A defect of OX6+ area may suggest disappearance of Ia+ epithelial reticular cells. This may be considered to represent an increase of immature thymocytes in FK506-treated thymus because the epithelial reticular cells in the thymus may contribute to thymocyte maturation (Farr et al., 1985). In addition to these features, some thymocytes in this area showed karyopyknosis, suggesting widespread cell death was occurring in this area. Such observations are similar to a report ofincreased macrophage phagocytic activity in the cortex in FK506-treated rats (Stephen et al., 1989). FK506-treated thymocytes showed low 3H-thymidine uptake compared with controls (Fig. 8). This suggests that the decrease of thymocytes at the S phase caused by FK506 interferes with thymocyte proliferation. However, the localization of FITC-labelled thymocytes in treated rats was not significantly different from that in normal rats. In addition, the atrophy of the thymus-dependent area was not remarkable in the spleen during FK506 treatment. These
observations may suggest that FK506 inhibits differentiation in the thymus, but does not interfere with mobilization of thymocytes. Following the cessation of FK506 treatment, medullary regions reformed. The reformed thymic medulla was similar to that of the control thymus in the expressions of antigens detected by several MoAbs such as OX6 and OX 18. These results confirm that the effect of FK506 on the thymus is reversible and that the altered lymphocyte generation kinetics occurring with treatment may be restored to those of normal rats. REFERENCES BRIDEAU, R.J., CARTER, P.B., MCMASTER, W.R., MASON, D.W. & WILLIAMS, A. F. (1980) Two subsets of rat T lymphocytes defined with monoclonal antibodies. Eur. J. Immunol. 10, 609. BUTCHER, E.C. & WEISSMAN, I.L. (1980) Direct fluorescent labeling of cells with fluorescein or rhodamine isothiocyanate. I. Technical aspects. J. immunol. Methods, 37, 97. COLLIER, D.S.J., CALNE, R., THIRU, S., FRIEND, P.J., LIM, S., WHITE, D.J.G., KOHNO, H. & LEVICKIS, J. (1988) FK506 in experimental renal allografts in dogs and primates. Transplant. Proc. 20, 226. FARR, A.G., ANDERSON, S.K., MARRACK, P. & KAPPLER, J. (1985) Expression of antigen-specific, major histocompatibility complexrestricted receptors by cortical and medullary thymocytes in situ. Cell, 43, 543. FUKUMOTO, T., MCMASTER, W.R. & WILLIAMS, A.F. (1982) Mouse monoclonal antibodies against rat major histocompatibility antigens. Two Ia antigens and expression of Ia and class I antigens in rat thymus. Eur. J. Immunol. 12, 237. JEFFERIES, W.A., GREEN, J.R. & WILLIAMS, A.F. (1985) Authentic T helper CD4(W3/25) antigen on rat peritoneal macrophages. J. evp. Med. 162, 117. MASON, D.W. & WILLIAMS, A.F. (1980) The kinetics of antibody binding to membrane antigens in solution and at the cell surface. Biochem. J. 187, 1. MASON, D.W., ARTHUR, R.P., DALLMAN, M.J., GREEN, J.R., SPICKETT, G.P. & THOMAS, M.L. (1983) Functions of rat T-lymphocyte subsets isolated by means of monoclonal antibodies. Immunol. Rev. 74, 426. MCMASTER, W.R. & WILLIAMS, A.F. (1979) Identification of Ia glycoproteins in rat thymus and purification from rat spleen. Eur. J. Immunol. 99, 426. NALESNIK, M.A., TODO, S., MURASE, N., GRYZAN, S., LEE, P.-H., MAKOWKA, L. & STARZL, T.E. (1987) Toxicology of FK-506 in the Lewis rat. Transplant. Proc. 19, 89. OCHIAI, T., NAKAJIMA, K., NAGATA, M., SUZUKI, T., ASANO, T., UEMATSU, T., GOTO, T., HORI, S., KENMOCHI, T., NAKAGORI, T. & ISINO K. (1987) Effect of a new immunosuppressive agent, FK506, on heterotopic cardiac allotransplantation in the rat. Transplant. Proc. 19, 1284. SAWADA, S., SUZUKI, G., KAWASE, Y. & TAKAKU, F. (1987) Novel immunosuppressive agent, FK506. In vitro effects on the cloned T cell activation. J. Immunol. 139, 1797. STEPHEN, M., Woo, J., HASAN, N.U., WHITING, P.H. & THOMSON, A.W. (1989) Immunosuppressive activity, lymphocyte subset analysis, and acute toxicity of FK-506 in the rat. Transplantation, 47, 60. TANAKA, M., SHINOHARA, K., FUKUMOTO, T., TANAKA, H. & KANEKO, T. (1988) Effect of cyclosporin A on rat thymus: time course analysis by immunoperoxodase technique and flow cytofluorometry. Clin. exp. Immunol. 72, 216. WILLIAMS, A.F., GALFRE, G. & MILSTEIN, C. (1977) Analysis of cell surfaces by xenogeneic myeloma-hybrid antibodies: differentiation antigens of rat lymphocytes. Cell, 12, 663. WILLIAMS, A.F. (1982) Surface molecules and cell interactions. J. theor. Biol. 98, 221.
Effects of FK506 on rat thymus: time-course analysis by immunoperoxidase technique and flow cytofluorometry K. TAKAI, K. JOJIMA, J. SAKATOKU & T. FUKUMOTO* Department of Urology and * First Department of Anatomy, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan
(Acceptedfor publication 4 July 1990)
SUMMARY The effect of administration of FK506 at 1 mg/kg body weight for 14 days on rat lymphoid tissues, especially the thymus, and recovery after discontinuation of treatment, were investigated by the immunoperoxidase technique and flow cytofluorometry using monoclonal antibodies OX6, OX7, OX8, OX 18 and W3/25, reactive with rat lymphocytes. Marked reduction of the thymic medulla upon treatment was clearly demonstrated by staining with OX 18 and OX6. Changes produced by FK506 were also observed in the cortical area of the thymus, and were especially marked in the subcapsular area and around blood vessels. Eventually, the thymic cortex appeared patchy, this change being maximal 14 days after the start of administration. Obvious restitution of the thymic medulla was evident about 14 days after withdrawal of FK506. Flow cytometric analysis of the thymus showed that the percentages of cells labelled positively with OX7, OX8 and W3/25 were increased with FK506 treatment, and recovered to the normal levels soon after withdrawal. Furthermore, the peak of fluorescence intensity of OX7+, OX8+ and W3/25+ cells showed a temporary shift to the right during FK506 treatment; however, the peak of fluorescence intensity of OX 18 + cells showed a temporary shift to the left. Treatment with FK506 also produced a significant change in 3H-thymidine uptake by thymocyte. These results suggest that FK506 may inhibit the proliferation, maturation and differentiation of thymocytes. However, thymocytes prepared from FK506-treated rats and labelled with FITC behaved similarly to rat thymocytes in normal recipient rats. This suggests that during FK506 treatment thymocytes may retain their potential for peripheral mobilization.
Keywords FK506 thymus monoclonal antibodies immunohistochemistry flow cytofluorometry
INTRODUCTION FK506 (formerly designated FR900506) was isolated from Streptomyces tukubaensis strain 9993 in the laboratories of Fujisawa Pharmaceutical Co. (Osaka, Japan) in 1984. It has a macrolide chemical structure with a molecular weight of 822. In vitro experiments have demonstrated that the agent inhibits interleukin-2 (IL-2) production and generation of cytotoxic T cells at a concentration 100 times lower than cyclosporin A (CyA) (Sawada et al., 1987). In vivo studies on the effects of FK506 in organ transplantation have demonstrated a remarkable immunosuppressive potency and prolongation of allograft survival time in rats with heart and renal transplants (Ochiai et al., 1987; Collier et al., 1987). In relation to the toxicological and histopathological effects of FK506, one study has shown lymphocytic depletion in Correspondence: K. Takai, Department of Urology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan.
445
the rat thymic cortex (Nalesnik et al., 1987). According to subsequent studies, however, a striking reduction in the size of the thymic medulla with increased macrophage phagocytic activity in the cortex has been observed after FK506 treatment in rats (Stephen et al., 1989). T cells develop in the thymus and may mature as they migrate from the cortex to the medulla and then to the periphery, acquiring OX18 antigen and selectively losing and acquiring W3/25 and OX8 antigens. Monoclonal antibodies (MoAbs) against these surface antigens have been useful for distinguishing thymocyte subpopulations in rats (Mason et al., 1983). We have recently analysed the effect of CyA on the rat thymus, using the immunoperoxidase technique and flow cytofluorometry (Tanaka et al., 1988). Here, using a similar technique, we investigated in more detail the effect of FK506 on the thymus and its recovery after withdrawal of FK506, using MoAbs. We also analysed the effect of FK506 on spleen in relation to effect in the thymus, using flow cytofluorometric
K. Takai et al.
446
analysis of thymus. We present the results here and discuss the effect of FK506 on rat thymus tissue in relation to its
450
immunosuppressive activity.
400 -
w
E
MATERIALS AND METHODS Animals For analysis of the thymus and spleen, inbred male LEW (RT-1 ) rats, aged 10 weeks (mean body weight 275 g) were used. They were obtained from Seiwa Experimental Animals, Fukuoka, Japan, and were kept in our animal colony under routine laboratory conditions with free access to food and water.
c52 350 a)
E 300
2 _H250
200 L
FK506 administration The first day of administration was taken as day 0 of the experiment. LEW rats received FK506 (1 mg/kg body weight) by daily intramuscular injection for 14 days (days 0 to 13) into one of the four limbs, with daily rotation.
Experimental protocol On days 0, 3, 7, 10, 14, 21 and 28 the rats were killed by ether anaesthesia and tissues were removed for immunohistochemistry (thymus and spleen) and flow cytofluorometry (thymus). Antibodies MoAbs used for identifying rat lymphocyte surface antigens were W3/25 (CD4), T helper cells (Williams, Galfre & Milstein, 1977) and macrophages (Jefferies, Green & Williams, 1985); OX8 (CD8), T cytotoxic/suppressor cells (Brideau et al., 1980); OX18, class I MHC gene product (Fukumoto, McMaster & Williams, 1982); OX6, class II MHC gene product (McMaster & Williams, 1979); OX7, Thy-1.1 antigen (Mason & Williams, 1980). These specific MoAbs (from ascites fluid) were purchased from Sera Labs (Sussex, UK). FITC-conjugated sheep F(ab')2 anti-mouse IgG was also obtained from Sera Labs. Indirect immunohistochemical staining FK506-treated rats were killed under anaesthesia, and the thymus was frozen. Immunohistochemical staining of the thymus and spleen was performed as described previously (Fukumoto et al., 1982). Briefly, frozen sections of the thymus and spleen were reacted with each MoAb and further stained by the ABC method using a mouse IgG Vectastain ABC Kit (Vector Labs, Burlingame, CA), using the MoAb at 1/10 dilution in phosphate-buffered saline (PBS). Flow cytofluorometry Single-cell suspensions were obtained by pressing the thymus through a stainless steel mesh into RPMI 1640 (Flow Labs, North Ryde, Australia) supplemented with 5% heat-inactivated fetal bovine serum (Flow Labs). Erythrocytes were lysed with hypotonic ammonium chloride. Cells from the thymus were labelled with MoAb and FITC-conjugated secondary antibody, and analysed by flow cytofluorometry (EPICS-753; Coulter Electronic Laboratories, Hialea, FL) as described by Fukumoto et al., (1982). The labelled cells were then analysed. To clarify the fate of FITC-labelled thymocytes in normal recipient rats, single-cell suspensions of thymus were obtained as described above. Thymocytes were labelled with FITC using the method described by Butcher & Weissman (1980), then 1-5 x 108 FITC-labelled thymus cells from FK506-treated and
0
3
7
14 Time (days)
21
28
Fig. 1. Thymus weights of FK506-treated rats (m) or control rats (0) at various times during the experiment. The mean value of four thymus weights (±s.e.m.) is shown in each case. Control and FK506-treated groups were compared by Student's t-test. The decreases in mean thymus weight on days 14 and 28 are statistically significant: *PN-
1
thymus sections
of frozen
Immunoperoxidase staining patterns
from FK 506-treated rats labelled with
strongly
Note that
!
observed in the medulla (M) in controls.
with OX 1 8
0X6
or
were
Day O
(b),
4>
-.
Day 3
observed in the cortex
Day 7
a)
(C) in controls (a and c). Marked reduction of the thymic medulla (M)
C
were
the the
specific staining
observed with
thymic
OX 18
with
medulla and reduction of 0X6
thymic
remarkable
cortex
appeared patchy (d,
in
subcapsular
the
(e and f). Magnifications: a-d,
medulla had almost cortex was
19;
e
and
area
x
+
x
e,
day
on
area are
f).
and
around
47; f,
the medulla continued until
day
14
This
100
positivity for
treatment
evident, eventually The
reduction
blood
vessels
resulted in
| .-,'~I|.
is
(Fig. 2d).
On
patchy reduction
day 21,
of
the
-.---'-
..-;,
--
r; cot-,
.
Day 21 Day 28
,,
reduction of
thymic
0X6.
Day 14
,a)
(V)
|!
complete
OX18 and
J0 a)
190.
x
after cessation of treatment, the reconstituted
FK506
Atrophy of
completely atrophied by day 7, whereas
comparatively preserved.
showed normal
3 (b).
7
days
Fluorescence intensity
medulla
Additionally,
OX6+
area
in
Fig. 5. Fluorescence profiles of thymocytes labelled with OX8 in FK506treated rat thymus.
the cortex (Fig. 2d, 2e, 2f). The reduction was especially remarkable in the subcapsular area and around blood vessels (Fig. 2e, 2f).
[
0
-T
90 [
Flow-cytofluorometric analysis of thymus By scatter analysis, thymocyte size was invariable among the samples from day 0 to day 28. The percentages of cells positively labelled with OX7, OX8 and W3/25 increased with FK506 treatment, and recovered to
4-
a1)
a)
80p 0
the normal levels 70
[
87-3 +0-8, 91-4 + 2-9, 96-1 +0 7, 74-5 + 3-0 and 78-4+ 8-3, respectively, compared with 75-7 + 5-3 for controls (day 0). The percentages (±s.d.) of OX8+ cells on days 3, 7, 14, 21 and 28 were 79 9 + 2-5, 89 9 + 1 9, 94-6 + 2-2, 83-8 + 2-1 and 75-6 + 5-0, respectively, compared with 72-4 + 4 9 for controls (day 0). The percentages (±s.d.) of W3/25+ cells on days 3, 7, 14, 21 and 28 were 78-2 + 1-5, 87-5 +0-8, 91-7 +±40, 73-2 + 5 2 and 69 8 +0 6, respectively, compared with 74-6 + 3 -7 for controls (day 0). were
0
3
7
14
21
28
Time (day)
Fig. 3. Immunophenotypic analysis of rat thymus. 1rhe percentages of positive cells labelled with OX7 (-), OX8 (0) and W'3/25 (0) increased with FK506 treatment, and recovered to the normail levels soon after FK506 withdrawal. The value for each normal rat tl hymus around this age were almost similar to those of day 0.
after drug withdrawal (Fig. 3). The OX7+ cells on days 3, 7, 14, 21 and 28
soon
percentages (±s.d.) of
448
K. Takai et al. re 'ah .
I\
P,. ,,,^ v Day 21
Day 28 Fluorescence intensity
Fig. 7. Fluorescence profiles of thymocytes labelled with OX18 in FK506-treated rat thymus.
The fluorescence profile of thymocytes from normal adult a wide range of fluorescence intensity. The peak of the fluorescence intensity was shifted to the right with FK506 treatment on day 3. The maximal shift was observed on day 14. However, after cessation of FK506, the peak shifted back to coincide with the peak of the control until day 28 (Fig. 4). Analysis of OX8 + thymocytes from normal adult rat showed that they constituted a major population. The peak of the fluorescence intensity was shifted to the right with FK506 treatment, and after withdrawal the peak shifted back to coincide with that of the control (Fig. 5). The fluorescence profiles of W3/25 in normal adult rats showed that most thymocytes were W3/25 +. The peak of the fluorescence intensity was shifted to the right with FK506 treatment, and after cessation the peak shifted back to coincide with that of the control (Fig. 6). About 40% of the thymocytes of normal adult rats were OX 18+ and showed a wide range of fluorescence intensity. The rats, stained with OX7, showed
percentage of positive cells labelled with OX 18 appeared not to change. However, FK506 treatment resulted in a decrease in the proportion of the brightly OX 18 positive cells (Fig. 7).
FITC-labelled thymocytes The percentages (± s.d.) of FITC-labelled thymocytes from FK506-treated rats in normal recipient spleen, thymus and cervical lymph nodes were 6-1 + 1-5, 0 7+0-1 and 1 4+0 2, respectively. The percentages of labelled thymocytes from control rats in normal recipient spleen, thymus and cervical lymph nodes were 5-1 + 1 0, 0 8 + 0 1 and 2-2 + 0 5, respectively. The localizations of the FITC-labelled thymocytes from FK506-treated and normal rats were not significantly different. Immunohistochemical analysis of spleen FK506 treatment resulted in slight reduction in number of OX8+ or W3/25+ cells in periarterial lymphocyte sheath of spleen. 3H-thymidine uptake FK506-treated thymocytes showed low 3H-thymidine uptake compared with controls (Fig. 8). DISCUSSION
The thymus is an important organ for immune responses, especially cellular immunity. Thymocytes proliferate and mature in the thymus. Cortical thymocytes are generally considered to be less mature and less differentiated than medullary thymocytes (Mason et al., 1983). Here we investigated the effect of FK506 on thymocytes after intramuscular injection of the drug. Treatment with FK506 produced the increases in the percentages of OX7 +, OX8 + and W3/25 + cells (Fig. 3). This was followed by rapid recovery after cessation of treatment. This
449
Effects of FK506 on rat thymus increase of OX8+ and W3/25+ thymocytes in the thymus suggests that double-positive thymocytes may increase in the thymus during FK506 treatment. A relative increase of brightly fluorescent OX7 + cells during FK506 treatment was observed by flow cytofluorometry (Fig. 4). This increase may suggest an increase of Thy-i antigen per thymocyte, since the size of the thymocytes during FK506 treatment did not change remarkably. Accordingly, this may in turn suggest an increase of immature thymocytes (Williams, 1982). On maturation, the expression of MHC class I antigen on thymocytes is increased in the thymus (Fukumoto et al., 1982). We observed a relative decrease of bright OX 18 + cells during FK506 treatment (Fig. 7), perhaps suggesting a decrease of class I antigens per thymocyte. Treatment with FK506 may interfere with cell maturation in the thymus, which would support the above conclusion, since the amount of MHC class I antigen per cell is thought to increase in parallel with thymocyte maturation (Fukumoto et al., 1982). On maturation, the surface antigens of thymocyte are considered to change from CD4-CD8- (double negative) to CD4+CD8+ (double positive) in the thymus, and then to change from CD4+CD8+ to CD4+CD8- or CD4-CD8+ (single positive) (Mason et al., 1983). Thus, it is possible to consider the double-positive thymocytes are the immature thymocytes. More than 80% of thymocytes of normal rats are usually double-positive immature types (Fukumoto et al., 1982). In the FK506-treated thymus (as shown in Fig. 3 and discussed) double-positive immature population were also the main population of the thymocytes. When these FK506-treated thymocytes, which are mostly double-positive types, were analysed by EPICS, we observed a relative increase of brightly fluorescent W3/25 + and OX8 + cells (Figs 5 and 6). These mean an increase of CD4 and CD8 antigen per double-positive thymocytes. These facts may suggest that in FK506-treated thymus there exist more immature thymocytes than in normal thymus, and FK506 interferes with thymocyte maturation. Treatment with FK506 resulted in marked reduction of the thymic medulla, which was examined by specific staining with OX18 and OX6 (Fig. 2b, 2d). Additionally, FK506 treatment resulted in marked reduction of the OX6+ area in the cortex (Fig. 2e, 2f). The reduction was especially remarkable in the subcapsular area and around blood vessels, and eventually the thymic cortex appeared patchy. Thus, the OX6- area increased during FK506 treatment. A defect of OX6+ area may suggest disappearance of Ia+ epithelial reticular cells. This may be considered to represent an increase of immature thymocytes in FK506-treated thymus because the epithelial reticular cells in the thymus may contribute to thymocyte maturation (Farr et al., 1985). In addition to these features, some thymocytes in this area showed karyopyknosis, suggesting widespread cell death was occurring in this area. Such observations are similar to a report ofincreased macrophage phagocytic activity in the cortex in FK506-treated rats (Stephen et al., 1989). FK506-treated thymocytes showed low 3H-thymidine uptake compared with controls (Fig. 8). This suggests that the decrease of thymocytes at the S phase caused by FK506 interferes with thymocyte proliferation. However, the localization of FITC-labelled thymocytes in treated rats was not significantly different from that in normal rats. In addition, the atrophy of the thymus-dependent area was not remarkable in the spleen during FK506 treatment. These
observations may suggest that FK506 inhibits differentiation in the thymus, but does not interfere with mobilization of thymocytes. Following the cessation of FK506 treatment, medullary regions reformed. The reformed thymic medulla was similar to that of the control thymus in the expressions of antigens detected by several MoAbs such as OX6 and OX 18. These results confirm that the effect of FK506 on the thymus is reversible and that the altered lymphocyte generation kinetics occurring with treatment may be restored to those of normal rats. REFERENCES BRIDEAU, R.J., CARTER, P.B., MCMASTER, W.R., MASON, D.W. & WILLIAMS, A. F. (1980) Two subsets of rat T lymphocytes defined with monoclonal antibodies. Eur. J. Immunol. 10, 609. BUTCHER, E.C. & WEISSMAN, I.L. (1980) Direct fluorescent labeling of cells with fluorescein or rhodamine isothiocyanate. I. Technical aspects. J. immunol. Methods, 37, 97. COLLIER, D.S.J., CALNE, R., THIRU, S., FRIEND, P.J., LIM, S., WHITE, D.J.G., KOHNO, H. & LEVICKIS, J. (1988) FK506 in experimental renal allografts in dogs and primates. Transplant. Proc. 20, 226. FARR, A.G., ANDERSON, S.K., MARRACK, P. & KAPPLER, J. (1985) Expression of antigen-specific, major histocompatibility complexrestricted receptors by cortical and medullary thymocytes in situ. Cell, 43, 543. FUKUMOTO, T., MCMASTER, W.R. & WILLIAMS, A.F. (1982) Mouse monoclonal antibodies against rat major histocompatibility antigens. Two Ia antigens and expression of Ia and class I antigens in rat thymus. Eur. J. Immunol. 12, 237. JEFFERIES, W.A., GREEN, J.R. & WILLIAMS, A.F. (1985) Authentic T helper CD4(W3/25) antigen on rat peritoneal macrophages. J. evp. Med. 162, 117. MASON, D.W. & WILLIAMS, A.F. (1980) The kinetics of antibody binding to membrane antigens in solution and at the cell surface. Biochem. J. 187, 1. MASON, D.W., ARTHUR, R.P., DALLMAN, M.J., GREEN, J.R., SPICKETT, G.P. & THOMAS, M.L. (1983) Functions of rat T-lymphocyte subsets isolated by means of monoclonal antibodies. Immunol. Rev. 74, 426. MCMASTER, W.R. & WILLIAMS, A.F. (1979) Identification of Ia glycoproteins in rat thymus and purification from rat spleen. Eur. J. Immunol. 99, 426. NALESNIK, M.A., TODO, S., MURASE, N., GRYZAN, S., LEE, P.-H., MAKOWKA, L. & STARZL, T.E. (1987) Toxicology of FK-506 in the Lewis rat. Transplant. Proc. 19, 89. OCHIAI, T., NAKAJIMA, K., NAGATA, M., SUZUKI, T., ASANO, T., UEMATSU, T., GOTO, T., HORI, S., KENMOCHI, T., NAKAGORI, T. & ISINO K. (1987) Effect of a new immunosuppressive agent, FK506, on heterotopic cardiac allotransplantation in the rat. Transplant. Proc. 19, 1284. SAWADA, S., SUZUKI, G., KAWASE, Y. & TAKAKU, F. (1987) Novel immunosuppressive agent, FK506. In vitro effects on the cloned T cell activation. J. Immunol. 139, 1797. STEPHEN, M., Woo, J., HASAN, N.U., WHITING, P.H. & THOMSON, A.W. (1989) Immunosuppressive activity, lymphocyte subset analysis, and acute toxicity of FK-506 in the rat. Transplantation, 47, 60. TANAKA, M., SHINOHARA, K., FUKUMOTO, T., TANAKA, H. & KANEKO, T. (1988) Effect of cyclosporin A on rat thymus: time course analysis by immunoperoxodase technique and flow cytofluorometry. Clin. exp. Immunol. 72, 216. WILLIAMS, A.F., GALFRE, G. & MILSTEIN, C. (1977) Analysis of cell surfaces by xenogeneic myeloma-hybrid antibodies: differentiation antigens of rat lymphocytes. Cell, 12, 663. WILLIAMS, A.F. (1982) Surface molecules and cell interactions. J. theor. Biol. 98, 221.
