Suguru Matsui

Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. II. Inhibition of the production of IL-2 and gamma-IFN, but not B cell-stimulating factor 2.

The mechanism whereby FK506 inhibits immune responses was assessed in in vitro human studies. FK506 inhibited in a dose-dependent manner both interleukin 2 and gamma-interferon secretion of PBMC stimulated with PHA. Complete inhibition was obtained at the concentration of 0.25 nM of FK506 for IL-2 and 1 nM of FK506 for gamma-IFN production. Inhibition of 50% (IC50) was detected with 0.06 nM for IL-2 and 0.25 nM for gamma-IFN production. On the other hand, FK506 could not inhibit the B cell-stimulating factor 2 (BSF-2) production of PBMC, indicating the possibility that FK506 might spare the B cell function. Cloned T cells and cloned B cells, once activated, were scarcely affected by the agent; neither IL-2-driven proliferation of cloned T cells nor BSF-2-driven proliferation of cloned B cells was inhibited by FK506 at any concentration.

Effect of Cyclosporine on the Endocrine and Exocrine Pancreas in Kidney Transplant Recipients

In order to assess whether cyclosporine (CsA) affects the endocrine and exocrine pancreas, 105 patient courses comprised of 87 living related donor (LRD) and 18 cadaver donor (CAD) transplants treated with cyclosporine and prednisolone (Pred) were compared with the results of historical controls of 170 LRD and 10 CAD transplants treated with azathioprine (Az) and Pred. All of the recipients were followed for over 6 months after transplantation. There were no differences in age, sex, Broca index, family history, and preoperative evaluation on diabetic dispositions between the two treatment groups. The incidence of diabetes mellitus (DM) requiring insulin therapy was higher in CsA-treated recipients (18/105, 17.1%) than in Az-treated recipients (23/180, 12.8%; P less than 0.05), although both the daily Pred and cumulative doses of methylprednisolone (MP) at the onset of DM were significantly smaller in the CsA group than in the Az group (26.1 +/- 2.2 mg v 41.4 +/- 3.4 mg, P less than 0.01 and 3,086 +/- 626 mg v 7,133 +/- 1,129 mg, P less than 0.01, respectively). Diabetic patients with CsA showed higher levels of blood glucose (401 +/- 46 mg/dL), but lower amounts of urinary glucose (40 +/- 4.3 g/d) compared with patients treated with Az (239 +/- 31 mg/dL, and 61.4 +/- 4.6 g/d, respectively, P less than 0.05). In the CsA group, the onset of DM was related to high CsA plasma trough levels (greater than 350 ng/mL) in 23% of patients. Insulin could be withdrawn within 3 months in six of eight patients who had been converted from CsA to Az.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effects Of Perioperative Portal Venous Inoculation With Donor Lymphocytes On Renal Allograft Survival In The Rat: I. Specific Prolongation Of Donor Grafts And Suppressor Factor In The Serum

In order to investigate the in vivo functional role of the liver in the immune responses in organ transplantation, effects of perioperative portal venous p.v. administration of donor lymphocytes on renal allograft survival were tested in the rat kidney transplant model. Donor lymphocytes were prepared from BN (BN, RT-1n) or third-party DA (RT1a) rat spleens and lymph nodes and injected p.v. or intravenously to Lewis (LEW, RT-1l) hosts on the day of transplantation (day 0). Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.6 days (n = 10). Intravenous administration of 1 x 10(8) BN cells to LEW hosts on day 0 caused a slight, but not significant, prolongation of renal allograft survival (MST = 9.5 +/- 3.0 days, n = 13, NS), whereas portal venous inoculation of 1 x 10(8) BN cells on day 0 remarkably prolonged renal graft survival to 22.2 +/- 5.3 (n = 10, P less than 0.01). The prolongation of graft survival was antigen-specific; the administration of 1 x 10(8) DA cells p.v. to LEW hosts did not prolong the survival of BN renal grafts (MST = 7.4 +/- 0.8, n = 5). Spleen cells from p.v. treated LEW hosts 10 days after transplantation had no suppressor effect on the one-way MLC reaction of normal LEW responder cells toward donor BN or third-party DA stimulators. On the other hand, when serum from p.v.-treated LEW hosts was added to MLC at a concentration of 3 per cent of total volume, it suppressed the MLC reaction toward donor BN cells by 71.6 per cent, but not toward third-party DA stimulators (-8.5 per cent suppression, NS). Histological examination of p.v.-treated LEW hosts at 10 days after transplantation revealed that the liver had normal lobular architecture without expansion of portal tracts and infiltration of inflammatory cells. On the other hand, the transplanted kidney demonstrated a moderate mononuclear cell infiltration around the artery without an interstitial hemorrhage. Moreover, adoptive transfer of the serum from p.v.-treated LEW rats into the virgin secondary LEW hosts significantly prolonged the graft survival of BN kidneys from 7.8 days to 18.9 +/- 5.5 days (P less than 0.01), but not third-party DA graft survivals (MST = 7.5 +/- 0.6 days), indicating that an antigen-specific tolerogenic factor was released into the circulation through the process of allogeneic cells in the liver.

Effect of a new immunosuppressive agent, FK506, on human lymphocyte responses in vitro. I. Inhibition of expression of alloantigen-activated suppressor cells, as well as induction of alloreactivity.

The effect of FK506 on in vitro human lymphocyte responses was assessed in comparison with cyclosporine. FK506 suppressed, in a dose-dependent fashion, the lymphocyte response to stimulation with PHA and with alloantigens in primary mixed lymphocyte reactions at a 70-100-fold lower concentration than CsA--namely, 50% inhibition (IC50) was obtained with 8.6 nM FK506 and with 750 nM CsA in the PHA response, and with 0.21 nM FK506 and with 20 nM CsA in MLR. Allocytolytic T lymphocyte induction was also inhibited by FK506, whereas the ability of CTL to lyse targets was not affected by the agent, indicating that FK506 did not affect the recognition and binding of alloantigen by CTL. FK506 inhibited, in a dose-dependent fashion, both IL-2 receptor and transferrin receptor expression on the alloactivated lymphocytes--whereas this agent inhibited only incompletely both expression of both receptors on lymphocytes stimulated with PHA. Lymphocytes from primary MLR cultured in the presence of FK506 were tested for suppressor cell activity on day 8 of culture. FK506 did not allow for the expression of alloantigen-activated suppressor cells when used in a dose sufficient to inhibit CTL generation.

Characterization and analysis of soluble suppressor factor from early human decidual cells

To assess the role of decidual cells (DC) in the maintenance of pregnancy, immunosuppressive activity of culture supernatants from human DC were investigated. Dispersed DC suspensions from decidual tissue of early pregnancies were prepared by an enzyme digestion method using collagenase and DNase, and were enriched over 90 per cent without contamination of macrophages and lymphocytes in the fraction, with specific gravity between 1.033 and 1.044 (fraction 2 [Fr2] ) by a Percoll discontinuous density gradient method. The culture supernatants of Fr2 cells suppressed the responses of normal peripheral blood lymphocytes to PHA, MLR, and killer T cell generation at the 50 per cent concentration. To determine the mechanism of the immunosuppressive activity of the culture supernatants, the effect of the supernatants on interleukin-2 and gamma-interferon production, as well as IL-2 receptor expression, on PBL was investigated. The supernatants from 3 x 10(6)/ml of DC cells inhibited not only IL-2 and gamma-INF production, but also IL-2 receptor expression, compared with normal controls. The supernatants also suppressed immunoglobulin (IgG and IgM) production by pokeweed mitogen-stimulated B cells. To purify the suppressor factor from culture supernatants of DC, serum free culture supernatants of 3 x 10(6)/ml of DC, which showed 32 per cent of inhibitory activity on MLR, were applied to gel filtration. Fractions between mw 67,000 and 43,000 suppressed the MLR. These results suggest that DC from decidua of early pregnancy excrete an immunosuppressive factor with a molecular weight between 43,000 and 67,000 daltons.

Infiltrating ductal carcinoma developing within cystosarcoma phyllodes —A case report—

Malignancy in cystosarcoma phyllodes is uncommon and often confined to the stromal element. An extremely rare case of infiltrating ductal carcinoma developing within the stroma of cystosarcoma phyllodes is reported herein. A breast tumor with a diameter of 15 cm, which was diagnosed as cystosarcoma phyllodes, was excised from the right breast of a 47-year old woman. The histopathological examination revealed that hyperplastic ductal epithelial cells with dark cytoplasm and enlarged hyperchromatic nuclei were infiltrating the stroma. Thus, a diagnosis of ductal carcinoma within cystosarcoma was made. Subsequently, a standard radical mastectomy was performed. No recurrence or metastasis has been observed over the post-operative period of 5 years and 6 months.

The effects of perioperative portal venous inoculation with donor lymphocytes on renal allograft survival in the rat. II : Phenotypic and functional analyses of graft-infiltrating cells

Phenotype, donor-specific cytolytic activity, and helper activity to release cytokines of cells infiltrating within renal allografts of hosts rendered unresponsive by perioperative administration of donor lymphocytes via the portal vein (p.v.) were investigated in order to analyze the mechanism of prolongation of allograft survival. Graft-infiltrating cells (GIC) were obtained from Lewis (LEW, RT-1l) hosts inoculated perioperatively with 1 x 10(8) donor Brown-Norway (BN, RT-1n) lymphocytes p.v., a group that displays prolonged renal allograft survival (MST: 22.2 +/- 5.3 days, n = 10) compared with an uninoculated control group (MST: 7.8 +/- 0.6 days, n = 10, P less than 0.01). The percentages of cytotoxic/suppressor T cells (OX-8+) and Ia-positive cells (OX-6+) in GIC (23.1 +/- 4.4% and 9.0 +/- 2.0%, respectively) and in spleen cells (7.5 +/- 2.6% and 8.5 +/- 1.1%, respectively) from p.v.-inoculated LEW hosts on day 6 postgrafting were significantly lower than those of uninoculated control recipients (GIC: OX-8; 39.4 +/- 8.2%, OX-6; 23.0 +/- 1.9%. SP cell: OX-8; 21.6 +/- 9.9%, OX-6; 12.7 +/- 0.4%, P less than 0.05). Cytolytic activity of GIC from tolerant hosts on day 6 postgrafting toward donor blastoid lymphocytes was significantly decreased (19.0 +/- 1.2% at E/T = 50), compared with that from control allografts during ongoing rejection (51.5 +/- 5.3%, P less than 0.01). The amounts of in vitro cytokine production of GIC from tolerant hosts after mitogen stimulation were remarkably decreased (IL-2: 8.7 +/- 1.4 U/ml, IL-3: 15.4 +/- 0.6 U/ml, and BSF-2: 24.6 +/- 3.5 U/ml) than those of uninoculated control hosts during ongoing rejection (IL-2: 19.6 +/- 2.9 U/ml, IL-3: 22.2 +/- 2.7 U/ml, and BSF-2: 67.5 +/- 13.2 U/ml, P less than 0.05). These results demonstrated that activation of both Tc cells and Th cells was inhibited in the spleen and in situ in renal allografts following administration of donor lymphocytes through the portal vein.

The in vivo immunosuppressive action of suppressor cells from alloantigen-cyclosporine-treated mice and the capacity of spleen cells to release interleukins and gamma-interferon.

Antigne-nonspecific suppressor T cells were identified in spleens of mice rendered unresponsive by sensitization of allogeneic antigen in combination with cyclosporine (CsA) treatement. Suppressor cells were obtained from C57BL/6 (B6, H-2b) mice treated with a single i.p. injection of 1 * 107 allogeneic P815 (H-2d) cells combined with a five-day course of CsA, a group that did not show any cytotoxic activity of spleen cells against P815 targets. These noncytolytic spleen cells displayed suppressor activity on the induction of cytotoxic T (Tc) cells of normal lymphocytes against not only P815 stimulator (80.9% suppression, P>0.01, responder:additional cell ratio = 0,5:1) but also thirdparty BW5147 (H-2k) stimulator (68.2% suppression, P>0.01). The unresponsive state appears to be due to suppressor T (Ts) cells that are nonadherent to plastic or nylon-wool, 1500 rads-sensitive, and Thy-1-positive. Capacitles of spleen cells from CsA-P815-treated mice to release cytokines (interleukin 1 [IL-1]), interleukin 2 [IL-2], interleukin 3 [IL-3], and -interferon [-IFN]) were examined. Spleen cells from CsA-P815-treated B6 mice displayed 84.1%, 91.7% and 90.8% inhibition (0.35pL0.07 U/ml, 1.4pL0.29 U/ml, and 7.0pL0.9 U/ml) of IL-1, Il-2, and -IFN production compared with normal mice (2.2pL0.54 U/ml, 16.9pL2.1 U/ml, and 76.0pL3.1 U/ml P<0.01), respectively. However, IL-3 production was significantly less inhibition (46.1%, 2.35pL1.0 U/ml in CsA-P815-treated mice and 4.36pL1.7 U/ml in normal mice) compared with other cytokines (IL-1, IL-2, -IFN). Two systems were employed to assess te immunosuppressive efficacy of antigen-nonspecific Ts cells in vivo. First, adoptive transfer (i.p.) of spleen cells harvested rom CsA-P815-treated mice ten days after treatment on 3 consecutive days (days 0, 1, 2) at a 3*107 cell dose into virgin B6 mice that were immunized with P815 cells (1*107, day 0) completely inhibited the development of Tc cells against P815 targets (5% specific cytolysis, effector:target ratio [E:T] = 200). The suppressor effect was immunologically nonspecific; adoptive transfer of Ts cells from CsA-P815-treated mice also abrogated the development of Tc cells against third=party Bw5147 cells. Second, introvenous injection of spleen cells (1*107) from CsA-P815=treated B6 mice on three consecutive days (days 0, 1, 2) abolished the capability of the B6 mice to develop delayed-type-hypersensitivity (DTH) responses, a sinduced by s.c.o, immunization, not only with DBa/2 also with AKR spleen cells. The suppressor T cells appeared to mediate its suppressor effects by soluble factor. The culture supernates of suppressor cells added to the mixed lymphocyte reaction between normal responder B6 spleen cells and irradiated p815 or BW5147 cells significantly inhibited the Tc cell induction against each stimulator (48.5% d 34.4% suppression compared with culture without suppressor factor, E:T=10) Thus, these studies demonstrated that both impairment of cytokine generation and induction and/or sparing of suppressor T cells medi8ate the unresponsive state induced by CsA.

Improved outcome of renal transplantation with cyclosporine compared with azathioprine —Experience in 33 recipients followed for over one year—

Long-term clinical aspects after kidney transplantation using cyclosporine (CsA) were studied in 33 patients who received kidney grafts from one haplotype identical living related donor and who were followed for at least one year. Both actual graft and patient survival rates were 97 per cent at one year. Incidence and severity of acute rejection were reduced to a greater extent in patients treated with CsA than in patients treated with azathioprine (AZ). The incidence of infections was low, and no serious bacterial infection occurred in these 33 patients. In 17 of 33 patients with a deteriorative graft function caused by intractable nephrotoxicity, CsA was converted to AZ. The mean serum creatinine level of converted patients was significantly higher than that of patients maintained with CsA at each time when a dose of CsA was stepwise reduced from 14 mg/kg/day to 6 mg/kg/day. Conversion to AZ improved graft function dramatically, although it resulted in reversible acute rejections in 4 patients. CsA induced hepatotoxicity occurred in 10 patients, but in all normal liver function was restored with decrease in the dose. The potent immunosuppressive effect of CsA compensates for its side effects. However, CsA should be converted to AZ when the chronic nephrotoxicity persists at a late stage of post-transplantation.

Prolongation of renal allograft survival in the rat treated with amniotic fluid.

To assess the role of amniotic fluid (AMF) in the maintenance of pregnancy, immunosuppressive effects of AMF were studied in vivo, and the mechanisms of suppressor activity were analyzed immunologically in vitro in the rat. Female Lewis (LEW, RT-1l) rats mated with Brown-Norway (BN, RT-1n) rats for 14 days were sacrificed and cell-free AMF was obtained. AMF was diafiltered with PBS (PH 7.2) and reconstituted to 2 OD units measured at 280 nm. Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.2 days (n = 10). Five days of intravenous inoculation of AMF into LEW hosts remarkably enhanced BN graft survivals (MST = 20.3 +/- 4.4 days, n = 12) compared with controls (P less than 0.01), and slightly prolonged third-party DA (RT-1a) graft survivals (MST = 9.4 +/- 0.8 days, n = 7) compared with control LEW hosts engrafted with a DA kidney (MST = 7.6 +/- 0.2 days, n = 6). Five days of intravenous inoculation of pregnant sera into LEW hosts had no effect on BN graft survival. The AMF suppressed the proliferative response of LEW lymphocytes against not only irradiated BN stimulator cells but also irradiated third-party DA stimulators. The AMF also suppressed allokiller T cell generation of normal LEW lymphocytes against BN cells by 70.1% and 51.3%, and against DA cells by 64.9% and 38.9% at concentrations of 25% and 12.5%, respectively (P less than 0.01). To dissect the immunosuppressive activity of AMF, the effect of AMF on cytokine production and interleukin 2 (IL-2) receptor expression of concanavalin A-stimulated lymphocytes were investigated. AMF suppressed interferon and IL-2 production. Interestingly, however, AMF did not suppress interleukin 3 (IL-3) and interleukin 6 (IL-6) production, as well as IL-2 receptor expression. These results demonstrated that rat AMF displayed a strong immunosuppression in vivo as well as in vitro, and that AMF might play an important role in the maintenance of pregnancy.