REGULATION OF LYMPHOCYTE BLASTOGENESIS AND ANTIBODY PRODUCTION BY SOLUBLE FACTOR RELEASED BY A HUMAN B-LYMPHOBLASTOID CELL LINE Keiko Morikawa,“*
Shigeru Morikawa,2 Katsuyuki Imai,3 Fumimaro Oseko’
Yam lB, a human B lymphoblastoid cell line, spontaneously produced an immunoregulatory factor, which suppresses blastogenesis and antibody formation by human lymphocytes. The Yam 1B cells, which were derived from the peripheral blood of an adult T-cell leukemia patient, have been established and maintained in our laboratory since 1985. This cell line expressed mature B-cell surface antigens including surface immunoglobulin M (IgM), CD23, and HLA-DR, had cytoplasmic IgM; and secreted small amounts of IgM in the culture supernatants. Yam 1B was positive for Epstein-Barr virus-associated antigen (EBNA) but negative for adult T-cell-associated antigen (ATLA). The serum-free Yam 1B culture supernatants (SN) inhibited the expression of transferrin R, but neither the expression of interleukin 2 (IL-2) R(CD25) nor the production of IL-2 in the lymphocytes stimulated with phytohemagglutin. Yam 1B SN also inhibited DNA synthesis by human T and B lymphocytes and immunoglobulin generation by normal B cells as well as by Epstein-Barr virustransformed human B lymphoblastoid cell lines. The inhibitory activity of Yam 1B SN was inactivated at 56°C and at pH 10 but was relatively stable at pH 2. It was abrogated by digestion with pronase and was partially stable by digestion with trypsin. Fractions collected from a Sephacryl S-300 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden) were found to have a peak of inhibitory activity of cell proliferation associated with molecules of apparent MW, of 43,000 to 67,000. The inhibitory activity of Yam 1B SN was not blocked by the anti-transforming growth factor B antibody. These results indicated that putative Yam 1B factor appeared to represent a novel immunoregulatory factor.
Copyright o 1991 by W.B. Saunders Company
Several nonspecific human soluble suppressor factors for lymphocyte proliferation or immunoglobulin production have been found in the culture supernatants of mitogen-activated and nonactivated peripheral blood mononuclear cells,1z2normal human T lymphocytes,3,4 and murine T-cell hybridoma,526 or of the macrophagelike cell line U 937 in mitogen-activated7 and nonactivated8.9 conditions. Though several of these activities have been proven to be derived from transforming growth factor-p (TGF-13),3,‘0 oncostatin M3,11 or interferon-y (IFN-Y),~ most of the factors remain less well-defined. TGF-l3 is one of the mononuclear cell-derived factors with such immunosuppressive activ-
ity. Recent studies have shown that normal B cells as well as Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines can secrete TGF-p.“,‘” In this study, we show that a B lymphoblastoid cell line, Yam lB, spontaneously secreted an inhibitory factor of the proliferative responses of human T and B lymphocytes. In addition, Yam 1B supernatants (SN) inhibited immunoglobulin generation by normal B cells and B-lymphoblastoid cell lines. The inhibitory activity was not abrogated with treatment by anti-TGF-l3 antibody. Yam 1B SN seemed to contain a novel inhibitory factor. RESULTS
From
the Departments of Internal Medicine,‘,* Pathology,’ and Biochemistry: Shimane Medical University, Izumo, Japan 693. *To whom reprint requests should be addressed. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0306-0008$5.00/O KEY
WORDS:
CYTOKINE,
B cell line/immunoregulation/human
Vol. 3, No. 6 (November),
1991: pp 609-618
Efect of Yam 1B Supernatants on Phytohemagglutin-Induced T-Cell Blastogenesis or Staphylococcus Aureus Cowan Z (SAC)-Induced B-Cell Blastogenesis The concentrated Yam 1B SN was examined for its effect on the proliferation of human T or B lympho609
610 / Morikawa
CYTOKINE,
eta1
cytes stimulated with phytohemagglutin (PHA-P l:l,OOO dilution), concanavalin A (ConA; 10 ng/mL), phorbol myristate acetate (PMA; 10 ng/mL) as T-cell mitogens or Staphylococcus Aureus Cowan I (SAC; 1:105 voli vol), PMA (10 ng/mL) as B-cell mitogens, respectively (Fig. 1). The Yam 1B SN remarkably suppressed the DNA synthesis of T and B cells stimulated with PMA as well as other types of mitogens. As the inhibitory effect of Yam 1B SN extended to both T and B lymphocytes, peripheral blood mononuclear cell (PBMC) was used as target cells for subsequent experiments unless designated otherwise. Time-Course Analysis of the Efsects of Yam 1B Supernatant Yam 1B SN was added at different times after incubation of PBMC with PHA-P to determine when the effect of Yam 1B SN occurred. The inhibitory effect of Yam 1B SN was pronounced even when the Yam 1B preparations were added 1 day after culture initiation with PHA stimulation (83% suppression). Yam 1B SN was still effective when added during the last 8 h of culture (60% suppression) compared with
B
Vol. 3, No. 6 (November
1991: 609-618)
the suppression when added at initiation (95% suppression) of the 3-day culture interval (data not shown). Yam 1B SN was not cytotoxic because the recovery and viability of human PBMC incubated with varying concentrations of Yam 1B SN for 3 days were not different from cells incubated in its absence by trypan blue dye exclusion test (data not shown). Effect of Yam 1B Supernatant Activation Antigens
on the Expression of
The sequential induction of interleukin 2 receptor (IL-2R) and transferrin R expression on the same cells is essential for DNA synthesis. Expressions of IL-2R and transferrin R on the cell surfaces are necessary in Gla to Glb transition and Glb to S transition in the cell cycle, respectively.‘4.‘5 To examine the effect of Yam 1B SN on the cell cycle, the expression of IL-2R and transferrin R on PBMC stimulated with PHA-P in the presence or absence of Yam 1B SN was analyzed by flow cytometry using indirect immunofluorescence methods (Fig. 2). A significant proportion of PHAstimulated PBMC expressed IL-2R and transferrin R on their cell surfaces. Treatment with Yam 1B SN did not interfere with the expression of IL-2R but decreased the transferrin R expression in PHA-stimulated PBMC during 72 h in culture. This result indicated that Yam 1B SN may act to inhibit the transition from the Glb to the S phase of the cell cycle. The Eflect of Yam 1B Supernatant T-Cell-Dependent Immunoglobulin
on Generation
Human PBMC was cultured in the presence of pokeweed mitogen (PWM) and varying concentrations of Yam 1B SN for 7 days at 5% CO, incubator. The addition of Yam 1B SN to the culture profoundly inhibited PWM-induced immunoglobulin generation by PBMC. Both IgG and IgM supernatant immunoglobulin levels were suppressed (Table 1). Effect of Yam 1B Supernatant on Interleukin Production by Phytohemagglutin-Stimulated Peripheral Blood Mononuclear Cells 0
10’
102
Reciprocal Figure 1. proliferation
Dose-response of tonsillar
10
1
& Dilution
0
103
102
10
1
of Yam 1B SN
curves of the effect T and B lymphocytes.
of Yam
1B SN on the
Highly purified T cells (A) or B cells (B) from tonsillar samples were cufiuieh with optimal co&entrationS oi the indicated mitogens (A) PHA (O), ConA (0), and PMA (A); and (B) PMA (0) and SAC (0) alone or in the presence of serum-free Yam 1B culture supernatants (SN; 50-fold concentration) as described in Materials and Methods. The cells were labeled with ?Hlthvmidine for the last 18 h of a 72-h culture period. Backgroukd pH]thymidine uptake without mitogens was less than 1,000 cpm in the direct assay. Vertical bars represent mean f SE of triplicate cultures.
2
PBMC was stimulated with PI54 in the presence of Yam 1B SN for 48 h and then IL-2 production of PBMC was examined. The addition of Yam 1B SN did not inhibit the IL-2 production of PHA-stimulated cells (Table 2). Effect of Yam 1B Supernatant Lines
on the Various Cell
When the effect of Yam 1B SN on the proliferation of a variety of cell types was examined, the growth of EBV-transformed B-lymphoblastoid lines (Yam 3B0 and 3Bl) and of a pre-T-cell line (HPB-MLp-W) was
Immunoregulatory
factor
produced
by a B-cell
line
/ 611
A
Fluorescence
Intensity
(log
10 units)
Figure 2. Flow cytometry analysis of the expression of IL-2R (CD23 PHA-stimulated PBMC in the presence of Yam 1B SN.
(A) and transferrin
R (B) of
PBMC was stimulated with PHA in the presence of Yam 1B SN (20% voI/vol) for 3 days. Flow cytometry analysis was performed by using a FACStar cell analyzer. The presence of IL-2R+ and transferrin R’ cells in each group was as follows: 9.6% and 7.6% in nonstimulated PBMC (-); 9% and 7.2% in PBMC cultured with Yam 1B SN (. .); 26% and 32% in PHA-stimulated PBMC (....,); and 36.8% and 13.1% in PHA/Yam lB-stimulated PBMC (- -). Three different experiments were performed and a representative result is presented.
suppressed by the addition of Yam 1B SN, but the proliferation of another EBV-transformed B-cell line (Daudi), myeloma line (ARH77), T-cell line (MOLT4) or myeloid cell line (K562) were not affected (data not shown). The data indicated that the Yam 1B inhibitory factor did not work specifically to a particular cell lineage. Next, Yam 1B SN was examined as to the effect of immunoglobulin secretion by EBV-transformed human B-lymphoblastoid cell lines, Yam 3Bl and HLNTABLE 1. Effect of Yam 1B supernatant on the immunoglohulin production in pokeweed-mitogen-driven system. Amount Cell source
PWM
-
PBMC
+
Tonsil
MNC
-
+
Yam 1B SN VJ)
0 0 3.1 6.2 12.5 25 0 0 3.1 6.2 12.5 25
W
463 8,667 2,734 1,491 1,135 1,144 101 2,762 2,195 1,339 1,414 1,112
Ith, which constitutively secrete IgM and IgG or IgA in their culture SN, respectively. Yam 1B SN completely inhibited IgG, IgM, and IgA production by these cell lines (data not shown). Physicochemical
Characterization
Next, we examined the nature of Yam 1B SN. Figure 3 shows the inhibitory effect of Yam 1B after treatment with heat, acid, alkali, or enzyme digestion. Concentrated Yam 1B SN without treatment completely inhibited PHA-induced blastogenesis in human PBMC at a 1:4 dilution. Alkali (pH 10.0) treatment for 6 h inactivated the suppressive activity of Yam 1B SN, but the suppressive activity was fully retained after
of Ig (ng/mL) W
207 2,881 600 310 169 98 249 1,294 1,477 1,241 567 446
Tonsillar MNC or PBMC (1 x 10’ cells per well) were cultured in the presence of PWM (150 dilution) for 7 days with or without Yam 1B culture SN. Amounts of immunoglobulin secreted in culture supernatants were determined by ELISA. The values represent the mean of triplicate cultures in one of three different experiments. MNC, mononuclear cells.
TABLE 2. Effect of Yam 1B SN on phytohemagglutinin-induced interleukin 2 production human peripheral blood mononuclear cells. Culture
Supernatants
Medium PHA PHA + Yam 3.1 6.2 12.5 25.0
(%)
in
[“H]Thymidine Incorporation in CTLL-II (cpm)
313 k 11 19,912 ? 843 1B SN 25,357 26,840 30,940 32,114
+ k k +-
716 412 991 701
Human PBMC was cultured at 1 x 10”imL in 24 microwells with PHA (1:lOO vol/vol) in the presence or absence of various concentrations of Yam 1B SN for 48 h. The supernatant was recovered by centrifugation. IL-2 activity in the supernatant was assayed at 10% vol/vol on an IL-2-dependent cell line CTLL-II as [‘Hlthymidine incorporation during the final 5 h of a 24 h culture.
612 / Morikawa et al.
CYTOKINE,
Treatment of Yam 1B SN
[3H] Thymidine 1
Incorporation 2
Vol. 3, No. 6 (November 1991: 609-618)
(cpm x 10a4) 3
4
Untreated PH
2.0
PH 10.0
Trypein
1
Protease
4v,
48hI
56*C,
1Omi.n
80°C,
1Omin
-20°C,
48hr
P I
-
properties
of Yam
Medium
Figure
3.
Physicochemical
1B SN.
Concentrated Yam 1B SN (50-fold) was tested by incubation at 56°C or 80°C for 10 min, incubated at 4°C or -20°C for 48 h; treated with 100 pg/mL of insoluble protease or insoluble trypsin at 37°C for 2 h; acidified to pH 2.0 or alkalized to pH 10.0.
exposure to acid (pH 2.0) for 6 h at 0°C. The inhibitory activity of Yam 1B SN was abrogated by digestion with pronase but was relatively stable to trypsin digestion, was inactivated by heat treatment (56°C SOOC),and was resistant to freezing-thawing.
Gel Filtrff tion When the concentrated crude materials were fractionated on a Sephacryl S-300 column (Pharmacia, Uppsala, Sweden) equilibrated with 20 mM Tris-HCL buffer, inhibitory activity for blastogenesis was eluted from the broad fractions (No. 35-40). These active fractions were then concentrated with ultrafiltration using Centriprep-10 and rechromatographed by the same column. Figure 4 showed the gel filtration profile and the inhibitory activity of the fractions from the column on blastogenesis of PHA-stimulated PBMC. The inhibitory activity was eluted in a relatively narrow peak, which corresponded to an MW, range of 43,000 to 67,000, the mean value for the peak of the activity being 48,500.
Anion ExchangeChromatography The active fractions were collected, concentrated, and dialyzed against 20 mM Tris-HCL buffer (pH 8.0) overnight and applied to a DEAE-Cellulofine column (Seikagaku Kogyo, Tokyo, Japan) equilibrated with the same buffer. After washing with the same buffer, each column was eluted with a salt gradient as indicated in Materials and Methods and assayed for the inhibitory activity. As shown in Fig. 5, a peak of the inhibitory activity was observed in the fraction eluted from the Tris-HCL buffer containing 0.02 M NaCl.
Neutralization of Fractionated Yam IB Factor With Anti-Transforming GrowthFactor p Antibody TGF-l3 refers to a family of polypeptides that is produced by a wide variety of cell types including platelets, lymphocytes, and monocytes.‘6 TGF-l3 has a potent suppressive activity on the proliferation of T cells”’ and B cells” as well as immunoglobulin production by normal B lymphocytes.12 As several biological
Immunoregulatoty
Ferritin
Figure umn
4.
Sephacryl
chromatography
S-300 cob
LOS
SSA
OA
factor
produced
by a B-cell
line
/ 613
cyt.c
Y2 2
of Yam
1B SN.
4.,-I E ti g E m,
The active fractions eluted from the initial Sephacryl S-300 gel filtration were collected, concentrated, and reapplied onto the same column that had been equilibrated with 20 mM TrisHCL buffer containing 0.3 M NaCL (pH 7.2). Each fraction was tested at 1:4 dilution for its effect on PHA-stimulated blastogenesis of PBMC (O-O) and assayed for protein content (O---O).
IO
40
Number
Fraction
activities of Yam 1B SN were very similar to those of TGF-P, we examined whether the inhibitory activity in the Yam 1B SN might derive from TGF-P. The fractionated Yam 1B material was preincubated with a rabbit antibody to TGF-l3 or a control IgG preparation being added to the culture of PHA-stimulated periph-
50
era1 blood lymphocytes. Pretreatment of the Yam 1B material did not block the inhibitory activity of Yam 1B SN, although this antibody neutralized TGF-l3 (Table 3). Because this polyclonal antibody has a capability to neutralize not only TGF-l3, but also TGF-l3, activity, the above data suggested that discrete components
,’
.’ .’
./’
Figure 5. DEAE-Cellulofine exchange chromatography Yam 1B SN.
/
A’ .5
9 ‘4 % b/ /’
/
,/‘I
/’
/
of
The active fractions eluted from Sephacryl S-300 gel filtration were collected and concentrated. After extensive dialysis against 20 mM Tris-HCL buffer (PH. 8.0) the material was applied to a DEAE-Cellulofine A-500 column equilibrated with the initial buffer. The column was eluted by a linear gradient of 20 mM Tris-HCL buffer containing 0 to 0.5 M NaCL (PH. 8.0) (-----). Each fraction was tested at 1:4 dilution for its effect on PHA-stimulated blastogenesis of PBMC (O-O) and assayed for protein content (O-0).
614
I Morikawa
et al.
CYTOKINE,
TABLE 3. Antibody to transforming growth factor p does not block inhibitory activity induced by Yam 1B supernatant. PHA
Yam 1B SN
Anti-TGF-P
TGF-P
+
-
-
-
+
+
-
-
+
+
+
-
+
-
-
+
-
-
+ + -
-
+ + -
+ + +
+
-
Lymphocyte [3H]Thymidine
35,638 2,159 2,696 34.297 ‘673 1,644 804 2,360 33,154 713
proliferation Uptake (cpm)
-c k 2 f 2 2 k k f k
2,028 416 344 2.043 lb0 295 126 132 3,780 136
PBMC was cultured for 3 days with or without PHA-P (I:100 dilution), Yam 1B SN (12.5% vol/vol), TGF-P (1 ng/mL), or anti-TGF-P (100 +g/mL) antibody. [iH]Thymidine (1 p,Ci/well) was added 18 h before harxsting on day 3. Values represent mean ? SEM of triplicate cultures.
from TGF-l3 in the Yam 1B SN contribute immune-suppressive activity.
to the
Bioassayof Transforming Growth Factor p Activity in Yam 1B Supernatant To examine the direct TGF-p-like biological activity in Yam 1B SN, partially purified Yam 1B SN by Sephacryl S-300 gel filtration was assayed for TGF-l3 biological activity. The Yam 1B SN had very low levels of TGF-P-like activity (0.04 ng/mL by growth inhibition assay). We sought to determine whether this concentration of TGF-B had the capacity to inhibit human lymphocyte mitogenesis. When human peripheral blood lymphocytes were stimulated with PHA-P in the presence or absence of natural or recombinant TGF-l3, preparations at concentrations of 0.001 to 1 ng/mL for 72 h and pulsed with [3H]thymidine at 18 h before harvesting, both TGF-l3 preparations had an inhibitory activity for lymphocyte mitogenesis at the concentration of 1 ng/mL, but no apparent suppression was detected at the concentrations of 0.001 to 0.1 ng/mL (data not shown). DISCUSSION Although the role of T lymphocytes and macrophages in releasing factors which inhibit the proliferative response of lymphocytes has been well documented,‘- the potential role of B lymphocytes in this process is not well established. This study showed that the human B-lymphoblastoid cell line Yam lB, which originated from the peripheral blood of an ATL patient, spontaneously produced an inhibitory factor of lymphocyte activation. Yam 1B SN inhibited the proliferative response to mitogens of both human T cells and B cells. The inhibition by Yam 1B SN was effective even when using
Vol. 3, No. 6 (November
1991: 609-618)
PMA, which directly stimulate protein kinase C in the lymphocyte activation pathway, as a mitogen for the proliferation. Time-course analysis showed that the suppression by Yam 1B SN was still effective even when added during the last 8 h of the 3-day culture interval. The Yam 1B SN appeared to inhibit the Glb to S progression of the cell growth phase, because it inhibited the expression of transferrin R, but not the expression of IL-2R(CD25) on PHA-stimulated human PBMC.14,15 Physicochemical studies showed that the Yam 1B SN was inactivated by heat treatment (56°C) sensitive to alkali (pH 10.0) relatively resistant to acid (pH 2.0) sensitive to pronase, and partly sensitive to trypsin treatment. We found the MW, of the Yam 1B inhibitory material was 43,000 to 67,000 as estimated by gel filtration. The Yam 1B material bound to DEAE-anion exchange chromatograph was eluted with Tris-HCL buffer containing 0.02 M NaCL. There are several suppressor factors derived from cell lines or normal mitogen-activated T cells. The macrophage lineage cell line U937 releases a suppressor factor.7-9 The U937 factor inhibits the mitogeninduced blastogenesis. Fujiwara et a1.8 found that it blocks IL-2R expression, but more recently Sugimura et a1.9 report the reverse. Physicochemically, it is sensitive to acid and alkali treatment and had an MW, of 85,000* or 67,000.9 Thus, this factor differs from the Yam 1B factors on the basis of these biological as well as physiocochemical properties. Supernatants from cultures of the B-lymphoblastoid cell line PGLC-33H contain a macrophage migration inhibitory factor (MIF).” The dissociated form of this MIF in the serum-free SN is associated with a suppressive activity for T-cell-dependent polyclonal immunoglobulin production by human PBMC, though it is unknown whether this MIF may directly inhibit immunoglobulin generation by B cells in the T-cellindependent pathway. Physicochemically, this material differs from Yam 1B factor because these MIF inhibitory factors are heat stable in 56°C and show two peaks of active fraction corresponding with appearent MW, of 60,000 and 20,000.‘8 Supernatants from activated human T lymphocytes contained a growth inhibitory factor for some cell lines or human PBMC.‘,3,4 Recent investigation by Brown et a1.3 showed that three growth inhibitory peptides, TGF-l3, IFN=y, and oncostatin M, are present in serum-free T-cellconditioned medium. Though the suppressive activities of these lymphokines appeared to resemble that of the Yam 1B SN, there are several differences in biological and physicochemical characteristics. The putative suppressive factor in Yam 1B SN differed from IFN-y. IFN-7 is purified under acidic conditions and with an
Immunoregulatory
MW, of 25,000 and 20,000. Although IFN-?/ inhibits the proliferation of Th2 murine helper T-lymphocyte clones,‘* it enhances the proliferative responses of mitogen-stimulated human B lymphocytes.‘9.20 Oncostatin M has also been isolated from serum-free supernatants of U937 cell lines.” Oncostatin M is stable at pH 2.0 and 11 and after heating for 1 h at 56oC.” Thus, Yam 1B differed from oncostatin M. Some, but not all, of the biological characteristics of Yam 1B SN resembled TGF-B.10,12Z13 TGF-B suppresses ConA-stimulated T-cell proliferation without interfering with the expression of IL-2R on ConAstimulated T cells and without blocking IL-2 synthesis21 as the Yam 1B SN did. Although the partially purified Yam 1B SN contained a very low level of TGF-B-like activity, which did not exhibit suppression on lymphocyte mitogenesis, Yam 1B SN had several characteristics that differ from TGF-l3. According to Kehrl et al., I2213TGF-B inhibits the proliferation and immunoglobulin secretion of human B cells, but not immunoglobulin production by an EBV-transformed B-cell line, CESS cells. Though we did not examine it by using CESS cells, Yam 1B SN suppressed immunoglobulin generation by EBV-transformed B-cell lines, Yam 3B1, and HLN-Ith cells (data not shown). The inhibitory activity of proliferative response induced by TGF-B was partly overcome by the addition of IL-2,12 but that induced by Yam 1B SN was not (data not shown). It has been reported that mitogen-activated human B lymphocytes release a very small quantity of TGF-13,12and that the biological activity by TGF-B on B lymphocytes is abrogated by the addition of an antibody specific for TGF-B,.13 Among TGF-B family, TGF-P,, TGF-B,, and TGF-B, are well studied.22 TGFpl, TGF-B,, and TGF-B, are equipotent in the degree to which they inhibit cell growth. TGF-B, is functionally similar to TGF-B, in its inhibition of B-cell proliferation.13 The polyclonal antibody for TGF-j3, shows the similar effect of neutralization of biological activities of both TGF-B, and TGF-j3,. The inhibitory activity of the Yam 1B factor was not abrogated after being neutralized by this anti-TGF-B antibody (Table 3). TGF-B is secreted by culture cells in an inactive or latent form in general. Several physicochemical treatments with acidification (less than pH 4), alkalinization (greater than pH 9),” or proteinases” will activate latent TGF-B. Proteolytic cleavage of TGF-B by the serine proteinase plasmin is relevant to physiological mechanism of activation.25 In contrast, Yam 1B inhibitory factor secreted by Yam 1B cells showed an activity without any treatment. Physicochemically, the activity of Yam 1B SN was abrogated by alkalinization (pH 10.0) or by pronase treatment. Activity of Yam 1B SN was not intensified with acidifacation but was slightly weakened. Crude TGF-l3 materials have been applied to a cation-exchange chromatography for purifica-
factor
produced
by a B-cell
line
/ 615
tionZ6 but the Yam 1B SN was not adsorbed in cation-exchange chromatography (data not shown). These results suggested that the inhibitory activity in the Yam 1B SN appeared to be unrelated to TGF-B activity. However, whether Yam 1B SN contains a new factor different from TGF-B remains unknown until the sequence of Yam 1B factor is obtained. Further purification and characterization of the inhibitory factors derived from Yam 1B is now under progress. Several other substances that inhibit lymphocyte function have been identified. For example, soluble immune suppressor supernatant (SISS)-B is produced by ConA-activated T lymphocytes. SISS-B has an MW, of 60,000 to 80,000, is stable at pH 2.5, and labile at 56°C although it has not been determined whether SISS-B inhibits B-cell proliferation. Soluble immune response suppressor (SIRS) and monoclonal nonspecific suppressor factor (MNSF) are murine suppressor factors that inhibit antibody secretion by B cells. These factors differ in MW, and sensitivity to heat and pH because they have a smaller MW, (14,000 to 24,000) than the suppressor fractions described here, are stable at 56°C and unstable at pH 2.0.5,6 A recent study by Lau et a1.27 shows that a suppressor-activating factor (SAF) produced by a mutant T-cell line is found to reside in a oxidized product of spermidine (SDA). Yam IB SN-derived factor appeared inconsistent with it being SDA because Yam 1B SN was prepared in serum-free medium after 3 days of culture. Yam IB SN is very sensitive to both heat and pronase treatment, which indicates that Yam IB SN is a protein. It has been reported that an EBV-transformed B-cell line secretes B-cell growth factors (BCGF) that sustain the proliferation in an autocrine 10op.~~Thus, it seems reasonable to assume that a similar autocrine inhibitory mechanism may function in cellular homeostasis as previously reported.” The putative Yam 1B factor may be one of a family of such autocrine growth inhibitory factors. It is interesting that Yam 1B cell line was derived from an ATL patient. It is well-known that ATL-T cells secrete several soluble growth factors; however, reports about B cells derived from ATL are very rare. It is known that the preferred target cells for HTLV-1, an etiologic agent of ATL, are CD4-expressing T cells, but B cells can be also infected with HTLV-1.29”3 The infected B cells are shown to have ATLA as well as EBNA29.32,33and IL-2R antigen (C~25).?9,30,32.33 Therefore, Yam 1B did not seem to be infected with HTLV-1. However, recent investigation showed that some infected B-cell lines have HTLV-1 clonally integrated without production of viral antigens.30,3’The integrated proviral DNA in the Yam 1B cell line is currently under investigation.
616 I Morikawa et al.
CYTOKINE,
MATERIALS AND METHODS Cell Lines Human B-lymphoblastoid cell line Yam 1B was originally derived from the peripheral blood of an adult T-cell leukemia (ATL) patient. The cell line has been established in our laboratory and has been maintained in RPM1 1640 supplemented with 10% fetal calf serum (FCS; Flow Laboratories, Mclean, VA) since 1985. The Yam 1B cell line showed common B-cell markers, including cell surface immunoglobulin (SIg), CD23, HLA-DR, and was negative for T cell (CD2, 3,4,8), myelomonocytic (CD13, 16), and CD25 (IL-2R, ~55) cell surface antigens. The cells possessed EBV-associated antigen (EBNA), but not adult T-cell-associated antigen (ATLA). In addition, the Yam 1B cell line had cytoplasmic IgM and spontaneously secreted small amounts of IgM (10 to 100 ng/mL) in the culture supernatants. EBV-transformed human B-cell lines, HLN-Ith and Yam 3B1, 3B0, and a pre-T-cell line, HPB-MLp-W, were also established in our laboratory and have been maintained in continuous culture for approximately 2 years. Other cell lines, Daudi, ARH 77, MOLT-4, and K 562, have been maintained in our laboratory for several years. All cell lines used in this experiment were mycoplasma-free on repeated testing.
Preparation
of Inhibitory
Factor Supernatants
For generation of inhibitory factor in serum-free medium, Yam 1B cells were washed and resuspended at 5 x 105/mL in serum-free RPM1 1640 medium at 37°C in a 5% CO, atmosphere. After 72 h of culture, cell-free supernatant fluids were harvested by centrifugation and were stored at -20°C until used. Serum-free Yam 1B SN were subsequently concentrated 50-700-fold for either assayof crude inhibitory factor activity or gel filtration under pressure using a ultrafiltration system with a PM 10 filter and with Centriprep-10 (Amicon Corp., Danvers, MA).
Gel Filtration Six milliliters of 700-fold concentrated Yam 1B SN was applied to a column (1.2 x 107 cm) of Sephacryl S-300 (Pharmacia), equilibrated with 20 mM Tris-HCL buffer containing 0.3 M NaCL (pH 7.2) at 4°C. Each fraction (2.3 mL) was filtered through 0.45 km membrane before being tested at a 1:4 dilution for effects on the cell proliferation. The inhibitory factor-containing fractions were collected, ultrafiltrated, and refractionated on the same columns. Rechromatographed fractions were retested for the inhibitory activity. The column was calibrated with cytochrome c(MW, 13,000), ovalbumin (MW, 43,000), bovine serum albumin (BSA: MW, 67,000), lactic dehydrogenese (LDH; MWr 140.000), and ferritin (MW, 440,000).
Anion-Exchange
Chromatography
The active fractions eluted from Sephacryl S-300 column were concentrated by ultrafiltration using Centriprep 10 and dialyzed extensively against 20 mM Tris-HCL buffer (pH 8.0) and applied to a DEAE-Cellulofine A-500 (Seikagaku Ko-
Vol. 3, No. 6 (November
1991: 609-618)
gyo) column equilibrated with the same buffer. After the column was washed with the starting buffer, the material was eluted with a linear gradient of 20 mM Tris-HCL buffer containing 0 to 0.5 M NaCL (PH. 8.0).
Protein Determination Protein concentration was determined either by using the Bio-Rad Laboratories protein assay or by measuring absorbance at 280 nm. Bovine serum albumin was used as a standard.
Cell Preparation Human tonsils were obtained by tonsillectomy from juvenile patients with chronic tonsillitis and were dispersed into single cell suspensions. Mononuclear cells were isolated on a Ficoll-Hypaque gradient. PBMCs were obtained from healthy donors. Monocytes and natural killer cells were depleted by incubation with 5 mM L-leucine methyl ester (Sigma, St. Louis, MO) in serum-free medium.34 T cellswere separated by rosetting twice with 2-aminoethylisothiouronium-bromide (Sigma)-treated sheep erythrocytes (E) and were obtained from rosetted (E+) cells by treating the attached E with lysing buffer [NH,CL:KHCO,] and then passing them over a nylon wool column. Nonrosetted (E-) cells were additionally purified by isolating small dense B cells from 60%/70% interface of a discontinuous percoll gradient (Pharmacia).
Assay for Inhibitory
Effect on Cell Proliferation
To test for inhibitory activity on the cell proliferation, 1 x lo5 cells were incubated with reciprocal dilutions of the concentrated Yam 1B SN preparations or control preparations and appropriate mitogens in RPM1 1640 supplemented with 10% FCS, L-glutamin, 2 mercaptoethanol, and penicillinstreptomycin. Cultures were stimulated with PHA-P; 1:lOO dilution (Difco Laboratories, Detroit, MI), ConA; 10 ng/mL (Pharmacia), phorbol myristate acetate (PMA); 10 ng/mL (Sigma), or SAC; 1:105 vol/vol (Calbiochem, Behring Co., CA). The cultures were incubated for 72 h in a 5% CO, incubator and pulse-labeled with 1 yCi [3H]thymidine for the final 18 h of culture. The cells were collected onto glassfilter paper using an automated cell harvester and the incorporated radioactivity was assessedby liquid scintillation counting. In some experiments, the effect of inhibitory factors on the proliferation of lymphoblastoid cell lines was determined by culturing 1 X lo5 cells in the presence of Yam 1B SN preparations or control preparations.
Assay for the Inhibitory Generation
Effect on Immunoglobulin
In assessingthe effect of Yam 1B SN on cell differentiation, PBMC was cultured at 1 x lo5 cellsin 200 PL containing RPM1 1640 with 10% FCS and varying concentrations of Yam 1B SN or control medium in the presence or absence of PWM (1:50 dilution; Gibco, Grand Island, NY) for 7 days. After that, the culture supernatants were harvested and the amount of immunoglobulin in the culture supernatants was determined by enzyme-linked immunosorbent assay(ELISA).
Immunoregulatory
factor produced by a B-cell line / 617
In some experiments, EBV-transformed human B-cell lines, which spontaneously secrete IgM and IgG (Yam 3Bl) and IgA (HLN-Ith), were used as target cells for evaluating the inhibitory effect of Yam 1B SN. The cell lines were cultured at 5 x lo4 cells per well in the presence or absence of Yam 1B SN for 4 days.
incubation at 4°C or -20°C for 48 h; heating at 56” or 80°C for 10 min; incubation at 37°C with and without insoluble protease (100 kg/mL; Sigma) or insoluble trypsin (100 kg/mL; Sigma) for 2 h; acidified to pH 2.0 with HCL or alkalized to pH 10.0 with NaOH for 6 h followed by adjustment to pH 7.2 and to the original volume.
Enzyme-Linked
Cytokines and Antibodies
Immunosorbent
Assay
Total immunoglobulin was determined by using ELISA as described in detail.3s In brief, 100 FL of the culture supernatants was added to microculture plates pre-coated with affinity-purified goat anti-human immunoglobulin (Cappel Laboratories, West Chester, PA). After incubation overnight at 4°C the microplates were washed and mixed with alkaline-phosphatase-conjugated anti-human IgM, IgG, and IgA (Cappel). After 2 h incubation at 37°C the plates were washed and the substrate paranitrophenylphosphate (Sigma) was added to each well. Absorbance at 405 nm was measured with Easy Reader EAR400 (SLT-Labinstruments, Austria). In each experiment, a standard curve was constructed from the samples containing a known concentration of immunoglobulin from normal human serum.
Immunojfuorescence For the detection of cell surface activation antigens, the cells were incubated with OKT9, a monoclonal antibody (MoAb) specific for transferrin R (Ortho Diagnostic Systems, Tokyo, Japan) or Tat-1 (anti-IL-2 R MoAb, CD25: donated by Dr. T. Uchiyama) for 20 min on ice, washed twice, and incubated with fluorescein isothiocyanate (FITC)labeled goat antibody to mouse IgG, F (ab’), (Cappel). Cell surface immunofluorescence was analyzed with FACScan (Becton-Dickinson, Mountain View, CA).
Phytohemagglutinin-Induced Production of Interleukin 2 in Human Peripheral Blood Lymphocytes Human PBMC was cultured at 1 X lo6 cells/mL (1 mL/well) in 24-well plates with or without Yam 1B SN in the presence or absence of PHA-P (1500 dilution) for 48 h at 37°C in a 5% CO, incubator. The culture medium consisted of RPM1 1640 with 1% FCS. The supernatants were recovered by centrifugation and were stored at -20°C until assay.
Assay of Interleukin
2 Activity
CTLL cells were washed extensively and were resuspended at 1 x lo5 cells/mL in the culture medium. One hundred microliters of the suspension was seeded in each well of flat-bottomed microtiter plate wells with 100 p,L of reciplocal dilution of sample supernatants. After 18 h of culture, 1 PC1 of [3H]thymidine was added to each well for an additional 6 h. The incorporated radioactivity was determined as described above for blastogenic response of PBMC.
Examination Supernatant Fifty-fold free condition
of Physicochemical
Nature of Yam 1B
concentrated Yam 1B SN produced in serumwas subjected to the following treatment:
Human recombinant and natural TGF-l3 were donated by Dr. T. Ikeda (King Brewing Co., Kakogawa, Japan). TGF+ neutralizing antibody was obtained from R & D Systems (Minneapolis, MN). Anti-TGF-l3 antibody was prepared from highly specific IgG fraction of rabbits immunized with highly purified, native porcine platelet TGF-l3. The antibody has been found to have a high titer for neutralization of the biological activities of TGF-l3, and TGF-6,. The antibody does not show any cross-reactivity with acidic or basic fibroblast growth factors, platelet-derived growth factor, or epidermal growth factor. Human IL-2 was purchased from Boehringer (Mannheim, Germany). IL-2 was produced by stimulating normal human T cells with PHA, and the culture supernatants were purified by using a sequence of ion-exchange chromatography and gel filtration. The resulting preparation was free of interferon and mitogenic concentrations of PHA (PHA concentration < 1 ng/mL).
Antibody Treatment The inhibitory factor-containing culture SN was preincubated with a previously determined optimal concentration of a rabbit antibody that neutralizes 1 p,g/mL of TGF-l3 (50 kg/mL) for 2 h at 4°C. Equivalent concentrations of normal rabbit IgG (Cappels) were added to parallel aliquots of SN as a control.
Transforming
Growth Factor p Assays
Supernatants to be assayed for TGF-l3 were obtained from partially purified Yam 1B SN by Sephacryl S-300 gel filtration. The supernatant was dialyzed with 0.2 M acetic acid before being assayed for TGF-l3. The bioassay for TGF-l3 depends on the growth inhibition of Mv 1 Lu mink lung epithelial cells (Ccl-64; American Type Culture Collection) measured by the [‘251]IdU incorporation into the DNA of actively growing cells after 72-h culture at 37°C in 5% CO, as previously described.36
Acknowledgment
The authors thank Dr. Tatsuhiko Ikeda for TGF-P assay and Akiko Kawakami for preparation of the manuscript. REFERENCES 1. Fleisher TA, Greene WC, Blaese RM, Waldmann TA (1981) Soluble suppressor supernatants elaborated by concanavarin A-activated human mononuclear cells. II. Characterization of a soluble suppressor of B cell immunoglobulin production. J Immunol 126:1192-1197.
618 I Morikawa
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et al.
2. Shou L, Schwartz SA, Good RA, Peng R, Chen human soluble suppressor factor affecting lymphocyte vitro. Proc Nat1 Acad Sci USA 77:6096-6100.
CL (1980) responses
A in
3. Brown TJ, Lioubin MN, Marquardt H (1987) Purification and characterization of cytostatic lymphokines produced by activated human T lymphocytes. J lmmunol 139:2977-2983. 4. Wilkins JA, Jordan Y, Rutherford WJ, Warrington RJ (1983) T cell factor-mediated suppression of immunoglobulin secretion by a human B lymphoblastoid cell line. J lmmunol 131:298-302. 5. Aune TM, Webb DR, Pierce CW (1983) Purification and initial characterization of the lymphokine soluble immune response suppressor. J lmmunol 131:2848-2852. 6. Nakamura characterization (MNSF) produced
of
M, Ogawa H, Tsunematsu T (1986) Isolation and a monoclonal nonspecific suppressor factor by a T cell hybridoma. J lmmunol136:2904-2909.
7. Wilkins JA, Sigurdson SL, Jordan Y, Ritherford WJ, Warrington RJ (1983) The production of immunoregulatory factors by a human macrophage-like cell line. 1. Characterization of an inhibitor of lymphocyte DNA synthesis. Cell lmmunol75:328-336. 8. Fujiwara H, Toossi 2, Oonishi K, Edmonds K, Ellner JJ (1987) Spontaneous production of a suppressor factor by a human macrophage-like cell line U937. Il. Suppression of antigenand mitogen-induced blastogenesis, IL 2 production and IL 2 receptor expression in T lymphocytes. J lmmunol 138:197-203. 9. Sugimura K, Ueda Y, Takeda K, Fukuda S, Tsukahara K, Habu Y, Fujiwara H, Azuma 1 (1989) A cytokine, lymphocyte blastogenesis inhibitory factor (LBIF), arrests mitogen-stimulated T lymphocytes at early Gl phase with no influence on interleukin 2 production and interleukin 2 receptor light chain expression. Eur J lmmunol 19:1357-1364. 10. Kehrl JH, Wakefield LM, Roberts AB, Jakowlew S, AlvarezMon M, Derynck R, Sporn MB, Fauci AS (1986) The production of TGF-P by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 163:1037-1050. 11. Zarling JM, Shoyab M, Marquardt H, Hanson MB, Lioubin MN, Todaro GJ (1986) Oncostatin M: A growth regulator produced by differentiated histiocytic lymphoma cells. Proc Nat1 Acad Sci USA 83:9739-9743. 12. Kehrl JH, Roberts AB, Wakefield LM, Jakowlew S, Sporn MB, Fauci AS (1986) Transforming growth factor p is an important immunomodulatory protein for human B lymphocytes. J lmmunol 137:3855-3860. 13. Kehrl JH, Taylor AS, Delsing GA, Roberts AB, Sporn MB, Fauci AS (1989) Further studies of the role of transforming growth factor-p in human B cell function. J lmmunol 143:1868-1874. 14. Bettens F, Kristensen F, Walker C, Schwulera U, Bonnard GD, de Week AL (1984) Lymphokine regulation of activated (Gl) lymphocytes Il. glucocorticoid and anti-Tat-induced inhibition of human T lymphocyte proliferation. J lmmunol 132:261-265. 15. Necker LM, Cossman J (1983) Transferrin tion in mitogen-stimulated human T lymphocytes DNA synthesis and cell division and is regulated Proc Nat1 Acad Sci USA 80:3494-3498.
receptor inducis required for by interleukin.”
16. Robert AB, Anzano MA, Wakefield LM, Roche NS, Stern DF, Sporn MB (1985) Type p transforming growth factor: A bifunctional regulator of cellular growth. Proc Nat1 Acad Sci USA 82:119-123. 17. Warrington RJ, Rutherford WJ, Sauder PJ (1982) Soluble mediators produced by a human continuous B-lymphoblastoid cell line. Cell lmmunol73:159-168. 18. Gajewski IFN-7 in immune Th2 dut not Thl 140:4245-4252.
TF, Fitch FW (1988) Anti-proliferative effect of regulation. 1. IFN-y inhibits the proliferation of murine helper T lymphocytes clones. J lmmunol
19. Romagnani S, Giudizi MG, Biagiotti R, Almerigogna F, Mongari C, Maggi E, Liang C, Moretta L (1986) B cell growth factor activity of interferon-y. Recombinant human interferon-y promotes
proliferation 136:3513-3516.
of anti-u
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activated
human
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B lymphocytes.
J lmmunol
20. Morikawa K, Kubagawa H, Suzuki T, Cooper Recombinant interferon-a, -p, and -y enhance the response of human B cells. J lmmunol139:761-766.
MD (1987) proliferative
21. Wahl SM, Hunt DA, Wong HL, Dougherty S, Francis N, Wahl LM, Ellingsworth L, Schmidt JA, Hall AB, Sporn MB (1988) Transforming growth factor-p immunosuppressive agent that inhibit IL-l-dependent proliferation. J lmmunol 140:3026-3032.
McCartneyG, Roberts is a potent lymphocyte
22. Barnard JA, Lyons RM, Moses HL (1990) The cell biology of transforming growth factor p. Biochem Biophys Acta 1032:79-87. 23. Pircher R, Jullien P, Lawrence DA (1986) P-Transforming growth factor is stored in human blood platelets as a latent high molecular weight complex. Biochem Biophys Res Commun 136:3033. 24. Lyons RM, Keski-Oja J, Moses HL (1988) activation of latent transforming growth factor-p from conditioned medium. J Cell Biol 106:1659-1665.
Proteolytic fibroblast-
25. Sato Y, R&in DB (1988) Autocrine activities of basic fibroblast growth factor: Regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis. J Cell Biol 107:1199-1205. 26. Frolik CA, Dart LL, Meyers CA, Smith DM, Sporn MB (1983) Purification and initial characterization of a type p transforming growth factor from human placenta. Proc Nat1 Acad Sci USA 80:3676-3680. 27. Lau C, Stanojev D, Visconti V, Pang H, Krepinsky G, Grey A. Wang E. lshaaue A (19901 Purification. characterization. and st;ucturil elucidation of t‘he active moiety df the previously dalled “suppressor actiating factor (SAF).” Cell lmmunol 125:92-106. 28. Muraguchi A, Nishimoto H, Kawamura N, Hori A, Kishimoto T (1986) B cell-derived BCGF functions as autocrine growth factor(s) in normal and transformed B lymphocytes. J lmmunol 137:179-186. 29. Longo DL, Gelmann EP, Cossman J, Young RA, Gallo RC, O’Brien SJ, Matis LA (1984) Isolation of HTLV-transformed B-lymphocyte clone from a patient with HTLV-associated adult T-cell leukemia. Nature 310:505-507. 30. Mann DL, Clark J, Clark M, Reitz M, Popovic M, Franchini G, Trainor CD, Strong DM, Blattner WA, Gallo RC (1984) Identification of the human T cell lymphoma virus in B cell lines established from patients with adult T cell leukemia. J Clin Invest 74~56-62. 31. Ng VL, Rainer C, Oliver MR, Marsh J, Wood P, Reyes GR, McGrath MS (1989) IgM secreted by human T lymphoma virus-linfected and transformed human B cell clones recognize a 66. kilodalton host-encoded protein. J lmmunol 143:2501-2507. 32. Sugamura K, Fujii M, Kobayashi N, Sakitani M, Hatanaka M, Hinuma Y (1984) Retrovirus-induced expression of interleukin 2 receptors on cells of human B-cell lineage. Proc Nat1 Acad Sci USA 81:7441-7445. 33. Yamamoto N, Matsumoto T, Koyanagi Y, Tanaka Y, Hinuma Y (1982) Unique cell lines harbouring both Epstein-Barr virus and adult T-cell leukemia virus. established from leukemia patients. Nature 299:367-369. 34. Thiele D, Lipskey PE (1985) Modulation of human natural killer cell function by t-leucine methyl ester: Monocyte-dependent depletion from human peripheral blood mononuclear cells. J lmmu/ no1 134:786-793. ., 35. Volkman DJ, Lane HC, Fauci AS (1981) Antigen-induced in vitro antibody production in human: A model for B cell activation and immunomodulation. Proc Nat1 Acad Sci USA 78:2528-2531. 36. lkeda T, Lioubin MN, Marquardt H (1987) Human transforming growth factor type p2: Production by a prostatic adenocarcinoma cell line, purification, and initial characterization. Biochemistry 26:2406-2410.
Shigeru Morikawa,2 Katsuyuki Imai,3 Fumimaro Oseko’
Yam lB, a human B lymphoblastoid cell line, spontaneously produced an immunoregulatory factor, which suppresses blastogenesis and antibody formation by human lymphocytes. The Yam 1B cells, which were derived from the peripheral blood of an adult T-cell leukemia patient, have been established and maintained in our laboratory since 1985. This cell line expressed mature B-cell surface antigens including surface immunoglobulin M (IgM), CD23, and HLA-DR, had cytoplasmic IgM; and secreted small amounts of IgM in the culture supernatants. Yam 1B was positive for Epstein-Barr virus-associated antigen (EBNA) but negative for adult T-cell-associated antigen (ATLA). The serum-free Yam 1B culture supernatants (SN) inhibited the expression of transferrin R, but neither the expression of interleukin 2 (IL-2) R(CD25) nor the production of IL-2 in the lymphocytes stimulated with phytohemagglutin. Yam 1B SN also inhibited DNA synthesis by human T and B lymphocytes and immunoglobulin generation by normal B cells as well as by Epstein-Barr virustransformed human B lymphoblastoid cell lines. The inhibitory activity of Yam 1B SN was inactivated at 56°C and at pH 10 but was relatively stable at pH 2. It was abrogated by digestion with pronase and was partially stable by digestion with trypsin. Fractions collected from a Sephacryl S-300 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden) were found to have a peak of inhibitory activity of cell proliferation associated with molecules of apparent MW, of 43,000 to 67,000. The inhibitory activity of Yam 1B SN was not blocked by the anti-transforming growth factor B antibody. These results indicated that putative Yam 1B factor appeared to represent a novel immunoregulatory factor.
Copyright o 1991 by W.B. Saunders Company
Several nonspecific human soluble suppressor factors for lymphocyte proliferation or immunoglobulin production have been found in the culture supernatants of mitogen-activated and nonactivated peripheral blood mononuclear cells,1z2normal human T lymphocytes,3,4 and murine T-cell hybridoma,526 or of the macrophagelike cell line U 937 in mitogen-activated7 and nonactivated8.9 conditions. Though several of these activities have been proven to be derived from transforming growth factor-p (TGF-13),3,‘0 oncostatin M3,11 or interferon-y (IFN-Y),~ most of the factors remain less well-defined. TGF-l3 is one of the mononuclear cell-derived factors with such immunosuppressive activ-
ity. Recent studies have shown that normal B cells as well as Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines can secrete TGF-p.“,‘” In this study, we show that a B lymphoblastoid cell line, Yam lB, spontaneously secreted an inhibitory factor of the proliferative responses of human T and B lymphocytes. In addition, Yam 1B supernatants (SN) inhibited immunoglobulin generation by normal B cells and B-lymphoblastoid cell lines. The inhibitory activity was not abrogated with treatment by anti-TGF-l3 antibody. Yam 1B SN seemed to contain a novel inhibitory factor. RESULTS
From
the Departments of Internal Medicine,‘,* Pathology,’ and Biochemistry: Shimane Medical University, Izumo, Japan 693. *To whom reprint requests should be addressed. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0306-0008$5.00/O KEY
WORDS:
CYTOKINE,
B cell line/immunoregulation/human
Vol. 3, No. 6 (November),
1991: pp 609-618
Efect of Yam 1B Supernatants on Phytohemagglutin-Induced T-Cell Blastogenesis or Staphylococcus Aureus Cowan Z (SAC)-Induced B-Cell Blastogenesis The concentrated Yam 1B SN was examined for its effect on the proliferation of human T or B lympho609
610 / Morikawa
CYTOKINE,
eta1
cytes stimulated with phytohemagglutin (PHA-P l:l,OOO dilution), concanavalin A (ConA; 10 ng/mL), phorbol myristate acetate (PMA; 10 ng/mL) as T-cell mitogens or Staphylococcus Aureus Cowan I (SAC; 1:105 voli vol), PMA (10 ng/mL) as B-cell mitogens, respectively (Fig. 1). The Yam 1B SN remarkably suppressed the DNA synthesis of T and B cells stimulated with PMA as well as other types of mitogens. As the inhibitory effect of Yam 1B SN extended to both T and B lymphocytes, peripheral blood mononuclear cell (PBMC) was used as target cells for subsequent experiments unless designated otherwise. Time-Course Analysis of the Efsects of Yam 1B Supernatant Yam 1B SN was added at different times after incubation of PBMC with PHA-P to determine when the effect of Yam 1B SN occurred. The inhibitory effect of Yam 1B SN was pronounced even when the Yam 1B preparations were added 1 day after culture initiation with PHA stimulation (83% suppression). Yam 1B SN was still effective when added during the last 8 h of culture (60% suppression) compared with
B
Vol. 3, No. 6 (November
1991: 609-618)
the suppression when added at initiation (95% suppression) of the 3-day culture interval (data not shown). Yam 1B SN was not cytotoxic because the recovery and viability of human PBMC incubated with varying concentrations of Yam 1B SN for 3 days were not different from cells incubated in its absence by trypan blue dye exclusion test (data not shown). Effect of Yam 1B Supernatant Activation Antigens
on the Expression of
The sequential induction of interleukin 2 receptor (IL-2R) and transferrin R expression on the same cells is essential for DNA synthesis. Expressions of IL-2R and transferrin R on the cell surfaces are necessary in Gla to Glb transition and Glb to S transition in the cell cycle, respectively.‘4.‘5 To examine the effect of Yam 1B SN on the cell cycle, the expression of IL-2R and transferrin R on PBMC stimulated with PHA-P in the presence or absence of Yam 1B SN was analyzed by flow cytometry using indirect immunofluorescence methods (Fig. 2). A significant proportion of PHAstimulated PBMC expressed IL-2R and transferrin R on their cell surfaces. Treatment with Yam 1B SN did not interfere with the expression of IL-2R but decreased the transferrin R expression in PHA-stimulated PBMC during 72 h in culture. This result indicated that Yam 1B SN may act to inhibit the transition from the Glb to the S phase of the cell cycle. The Eflect of Yam 1B Supernatant T-Cell-Dependent Immunoglobulin
on Generation
Human PBMC was cultured in the presence of pokeweed mitogen (PWM) and varying concentrations of Yam 1B SN for 7 days at 5% CO, incubator. The addition of Yam 1B SN to the culture profoundly inhibited PWM-induced immunoglobulin generation by PBMC. Both IgG and IgM supernatant immunoglobulin levels were suppressed (Table 1). Effect of Yam 1B Supernatant on Interleukin Production by Phytohemagglutin-Stimulated Peripheral Blood Mononuclear Cells 0
10’
102
Reciprocal Figure 1. proliferation
Dose-response of tonsillar
10
1
& Dilution
0
103
102
10
1
of Yam 1B SN
curves of the effect T and B lymphocytes.
of Yam
1B SN on the
Highly purified T cells (A) or B cells (B) from tonsillar samples were cufiuieh with optimal co&entrationS oi the indicated mitogens (A) PHA (O), ConA (0), and PMA (A); and (B) PMA (0) and SAC (0) alone or in the presence of serum-free Yam 1B culture supernatants (SN; 50-fold concentration) as described in Materials and Methods. The cells were labeled with ?Hlthvmidine for the last 18 h of a 72-h culture period. Backgroukd pH]thymidine uptake without mitogens was less than 1,000 cpm in the direct assay. Vertical bars represent mean f SE of triplicate cultures.
2
PBMC was stimulated with PI54 in the presence of Yam 1B SN for 48 h and then IL-2 production of PBMC was examined. The addition of Yam 1B SN did not inhibit the IL-2 production of PHA-stimulated cells (Table 2). Effect of Yam 1B Supernatant Lines
on the Various Cell
When the effect of Yam 1B SN on the proliferation of a variety of cell types was examined, the growth of EBV-transformed B-lymphoblastoid lines (Yam 3B0 and 3Bl) and of a pre-T-cell line (HPB-MLp-W) was
Immunoregulatory
factor
produced
by a B-cell
line
/ 611
A
Fluorescence
Intensity
(log
10 units)
Figure 2. Flow cytometry analysis of the expression of IL-2R (CD23 PHA-stimulated PBMC in the presence of Yam 1B SN.
(A) and transferrin
R (B) of
PBMC was stimulated with PHA in the presence of Yam 1B SN (20% voI/vol) for 3 days. Flow cytometry analysis was performed by using a FACStar cell analyzer. The presence of IL-2R+ and transferrin R’ cells in each group was as follows: 9.6% and 7.6% in nonstimulated PBMC (-); 9% and 7.2% in PBMC cultured with Yam 1B SN (. .); 26% and 32% in PHA-stimulated PBMC (....,); and 36.8% and 13.1% in PHA/Yam lB-stimulated PBMC (- -). Three different experiments were performed and a representative result is presented.
suppressed by the addition of Yam 1B SN, but the proliferation of another EBV-transformed B-cell line (Daudi), myeloma line (ARH77), T-cell line (MOLT4) or myeloid cell line (K562) were not affected (data not shown). The data indicated that the Yam 1B inhibitory factor did not work specifically to a particular cell lineage. Next, Yam 1B SN was examined as to the effect of immunoglobulin secretion by EBV-transformed human B-lymphoblastoid cell lines, Yam 3Bl and HLNTABLE 1. Effect of Yam 1B supernatant on the immunoglohulin production in pokeweed-mitogen-driven system. Amount Cell source
PWM
-
PBMC
+
Tonsil
MNC
-
+
Yam 1B SN VJ)
0 0 3.1 6.2 12.5 25 0 0 3.1 6.2 12.5 25
W
463 8,667 2,734 1,491 1,135 1,144 101 2,762 2,195 1,339 1,414 1,112
Ith, which constitutively secrete IgM and IgG or IgA in their culture SN, respectively. Yam 1B SN completely inhibited IgG, IgM, and IgA production by these cell lines (data not shown). Physicochemical
Characterization
Next, we examined the nature of Yam 1B SN. Figure 3 shows the inhibitory effect of Yam 1B after treatment with heat, acid, alkali, or enzyme digestion. Concentrated Yam 1B SN without treatment completely inhibited PHA-induced blastogenesis in human PBMC at a 1:4 dilution. Alkali (pH 10.0) treatment for 6 h inactivated the suppressive activity of Yam 1B SN, but the suppressive activity was fully retained after
of Ig (ng/mL) W
207 2,881 600 310 169 98 249 1,294 1,477 1,241 567 446
Tonsillar MNC or PBMC (1 x 10’ cells per well) were cultured in the presence of PWM (150 dilution) for 7 days with or without Yam 1B culture SN. Amounts of immunoglobulin secreted in culture supernatants were determined by ELISA. The values represent the mean of triplicate cultures in one of three different experiments. MNC, mononuclear cells.
TABLE 2. Effect of Yam 1B SN on phytohemagglutinin-induced interleukin 2 production human peripheral blood mononuclear cells. Culture
Supernatants
Medium PHA PHA + Yam 3.1 6.2 12.5 25.0
(%)
in
[“H]Thymidine Incorporation in CTLL-II (cpm)
313 k 11 19,912 ? 843 1B SN 25,357 26,840 30,940 32,114
+ k k +-
716 412 991 701
Human PBMC was cultured at 1 x 10”imL in 24 microwells with PHA (1:lOO vol/vol) in the presence or absence of various concentrations of Yam 1B SN for 48 h. The supernatant was recovered by centrifugation. IL-2 activity in the supernatant was assayed at 10% vol/vol on an IL-2-dependent cell line CTLL-II as [‘Hlthymidine incorporation during the final 5 h of a 24 h culture.
612 / Morikawa et al.
CYTOKINE,
Treatment of Yam 1B SN
[3H] Thymidine 1
Incorporation 2
Vol. 3, No. 6 (November 1991: 609-618)
(cpm x 10a4) 3
4
Untreated PH
2.0
PH 10.0
Trypein
1
Protease
4v,
48hI
56*C,
1Omi.n
80°C,
1Omin
-20°C,
48hr
P I
-
properties
of Yam
Medium
Figure
3.
Physicochemical
1B SN.
Concentrated Yam 1B SN (50-fold) was tested by incubation at 56°C or 80°C for 10 min, incubated at 4°C or -20°C for 48 h; treated with 100 pg/mL of insoluble protease or insoluble trypsin at 37°C for 2 h; acidified to pH 2.0 or alkalized to pH 10.0.
exposure to acid (pH 2.0) for 6 h at 0°C. The inhibitory activity of Yam 1B SN was abrogated by digestion with pronase but was relatively stable to trypsin digestion, was inactivated by heat treatment (56°C SOOC),and was resistant to freezing-thawing.
Gel Filtrff tion When the concentrated crude materials were fractionated on a Sephacryl S-300 column (Pharmacia, Uppsala, Sweden) equilibrated with 20 mM Tris-HCL buffer, inhibitory activity for blastogenesis was eluted from the broad fractions (No. 35-40). These active fractions were then concentrated with ultrafiltration using Centriprep-10 and rechromatographed by the same column. Figure 4 showed the gel filtration profile and the inhibitory activity of the fractions from the column on blastogenesis of PHA-stimulated PBMC. The inhibitory activity was eluted in a relatively narrow peak, which corresponded to an MW, range of 43,000 to 67,000, the mean value for the peak of the activity being 48,500.
Anion ExchangeChromatography The active fractions were collected, concentrated, and dialyzed against 20 mM Tris-HCL buffer (pH 8.0) overnight and applied to a DEAE-Cellulofine column (Seikagaku Kogyo, Tokyo, Japan) equilibrated with the same buffer. After washing with the same buffer, each column was eluted with a salt gradient as indicated in Materials and Methods and assayed for the inhibitory activity. As shown in Fig. 5, a peak of the inhibitory activity was observed in the fraction eluted from the Tris-HCL buffer containing 0.02 M NaCl.
Neutralization of Fractionated Yam IB Factor With Anti-Transforming GrowthFactor p Antibody TGF-l3 refers to a family of polypeptides that is produced by a wide variety of cell types including platelets, lymphocytes, and monocytes.‘6 TGF-l3 has a potent suppressive activity on the proliferation of T cells”’ and B cells” as well as immunoglobulin production by normal B lymphocytes.12 As several biological
Immunoregulatoty
Ferritin
Figure umn
4.
Sephacryl
chromatography
S-300 cob
LOS
SSA
OA
factor
produced
by a B-cell
line
/ 613
cyt.c
Y2 2
of Yam
1B SN.
4.,-I E ti g E m,
The active fractions eluted from the initial Sephacryl S-300 gel filtration were collected, concentrated, and reapplied onto the same column that had been equilibrated with 20 mM TrisHCL buffer containing 0.3 M NaCL (pH 7.2). Each fraction was tested at 1:4 dilution for its effect on PHA-stimulated blastogenesis of PBMC (O-O) and assayed for protein content (O---O).
IO
40
Number
Fraction
activities of Yam 1B SN were very similar to those of TGF-P, we examined whether the inhibitory activity in the Yam 1B SN might derive from TGF-P. The fractionated Yam 1B material was preincubated with a rabbit antibody to TGF-l3 or a control IgG preparation being added to the culture of PHA-stimulated periph-
50
era1 blood lymphocytes. Pretreatment of the Yam 1B material did not block the inhibitory activity of Yam 1B SN, although this antibody neutralized TGF-l3 (Table 3). Because this polyclonal antibody has a capability to neutralize not only TGF-l3, but also TGF-l3, activity, the above data suggested that discrete components
,’
.’ .’
./’
Figure 5. DEAE-Cellulofine exchange chromatography Yam 1B SN.
/
A’ .5
9 ‘4 % b/ /’
/
,/‘I
/’
/
of
The active fractions eluted from Sephacryl S-300 gel filtration were collected and concentrated. After extensive dialysis against 20 mM Tris-HCL buffer (PH. 8.0) the material was applied to a DEAE-Cellulofine A-500 column equilibrated with the initial buffer. The column was eluted by a linear gradient of 20 mM Tris-HCL buffer containing 0 to 0.5 M NaCL (PH. 8.0) (-----). Each fraction was tested at 1:4 dilution for its effect on PHA-stimulated blastogenesis of PBMC (O-O) and assayed for protein content (O-0).
614
I Morikawa
et al.
CYTOKINE,
TABLE 3. Antibody to transforming growth factor p does not block inhibitory activity induced by Yam 1B supernatant. PHA
Yam 1B SN
Anti-TGF-P
TGF-P
+
-
-
-
+
+
-
-
+
+
+
-
+
-
-
+
-
-
+ + -
-
+ + -
+ + +
+
-
Lymphocyte [3H]Thymidine
35,638 2,159 2,696 34.297 ‘673 1,644 804 2,360 33,154 713
proliferation Uptake (cpm)
-c k 2 f 2 2 k k f k
2,028 416 344 2.043 lb0 295 126 132 3,780 136
PBMC was cultured for 3 days with or without PHA-P (I:100 dilution), Yam 1B SN (12.5% vol/vol), TGF-P (1 ng/mL), or anti-TGF-P (100 +g/mL) antibody. [iH]Thymidine (1 p,Ci/well) was added 18 h before harxsting on day 3. Values represent mean ? SEM of triplicate cultures.
from TGF-l3 in the Yam 1B SN contribute immune-suppressive activity.
to the
Bioassayof Transforming Growth Factor p Activity in Yam 1B Supernatant To examine the direct TGF-p-like biological activity in Yam 1B SN, partially purified Yam 1B SN by Sephacryl S-300 gel filtration was assayed for TGF-l3 biological activity. The Yam 1B SN had very low levels of TGF-P-like activity (0.04 ng/mL by growth inhibition assay). We sought to determine whether this concentration of TGF-B had the capacity to inhibit human lymphocyte mitogenesis. When human peripheral blood lymphocytes were stimulated with PHA-P in the presence or absence of natural or recombinant TGF-l3, preparations at concentrations of 0.001 to 1 ng/mL for 72 h and pulsed with [3H]thymidine at 18 h before harvesting, both TGF-l3 preparations had an inhibitory activity for lymphocyte mitogenesis at the concentration of 1 ng/mL, but no apparent suppression was detected at the concentrations of 0.001 to 0.1 ng/mL (data not shown). DISCUSSION Although the role of T lymphocytes and macrophages in releasing factors which inhibit the proliferative response of lymphocytes has been well documented,‘- the potential role of B lymphocytes in this process is not well established. This study showed that the human B-lymphoblastoid cell line Yam lB, which originated from the peripheral blood of an ATL patient, spontaneously produced an inhibitory factor of lymphocyte activation. Yam 1B SN inhibited the proliferative response to mitogens of both human T cells and B cells. The inhibition by Yam 1B SN was effective even when using
Vol. 3, No. 6 (November
1991: 609-618)
PMA, which directly stimulate protein kinase C in the lymphocyte activation pathway, as a mitogen for the proliferation. Time-course analysis showed that the suppression by Yam 1B SN was still effective even when added during the last 8 h of the 3-day culture interval. The Yam 1B SN appeared to inhibit the Glb to S progression of the cell growth phase, because it inhibited the expression of transferrin R, but not the expression of IL-2R(CD25) on PHA-stimulated human PBMC.14,15 Physicochemical studies showed that the Yam 1B SN was inactivated by heat treatment (56°C) sensitive to alkali (pH 10.0) relatively resistant to acid (pH 2.0) sensitive to pronase, and partly sensitive to trypsin treatment. We found the MW, of the Yam 1B inhibitory material was 43,000 to 67,000 as estimated by gel filtration. The Yam 1B material bound to DEAE-anion exchange chromatograph was eluted with Tris-HCL buffer containing 0.02 M NaCL. There are several suppressor factors derived from cell lines or normal mitogen-activated T cells. The macrophage lineage cell line U937 releases a suppressor factor.7-9 The U937 factor inhibits the mitogeninduced blastogenesis. Fujiwara et a1.8 found that it blocks IL-2R expression, but more recently Sugimura et a1.9 report the reverse. Physicochemically, it is sensitive to acid and alkali treatment and had an MW, of 85,000* or 67,000.9 Thus, this factor differs from the Yam 1B factors on the basis of these biological as well as physiocochemical properties. Supernatants from cultures of the B-lymphoblastoid cell line PGLC-33H contain a macrophage migration inhibitory factor (MIF).” The dissociated form of this MIF in the serum-free SN is associated with a suppressive activity for T-cell-dependent polyclonal immunoglobulin production by human PBMC, though it is unknown whether this MIF may directly inhibit immunoglobulin generation by B cells in the T-cellindependent pathway. Physicochemically, this material differs from Yam 1B factor because these MIF inhibitory factors are heat stable in 56°C and show two peaks of active fraction corresponding with appearent MW, of 60,000 and 20,000.‘8 Supernatants from activated human T lymphocytes contained a growth inhibitory factor for some cell lines or human PBMC.‘,3,4 Recent investigation by Brown et a1.3 showed that three growth inhibitory peptides, TGF-l3, IFN=y, and oncostatin M, are present in serum-free T-cellconditioned medium. Though the suppressive activities of these lymphokines appeared to resemble that of the Yam 1B SN, there are several differences in biological and physicochemical characteristics. The putative suppressive factor in Yam 1B SN differed from IFN-y. IFN-7 is purified under acidic conditions and with an
Immunoregulatory
MW, of 25,000 and 20,000. Although IFN-?/ inhibits the proliferation of Th2 murine helper T-lymphocyte clones,‘* it enhances the proliferative responses of mitogen-stimulated human B lymphocytes.‘9.20 Oncostatin M has also been isolated from serum-free supernatants of U937 cell lines.” Oncostatin M is stable at pH 2.0 and 11 and after heating for 1 h at 56oC.” Thus, Yam 1B differed from oncostatin M. Some, but not all, of the biological characteristics of Yam 1B SN resembled TGF-B.10,12Z13 TGF-B suppresses ConA-stimulated T-cell proliferation without interfering with the expression of IL-2R on ConAstimulated T cells and without blocking IL-2 synthesis21 as the Yam 1B SN did. Although the partially purified Yam 1B SN contained a very low level of TGF-B-like activity, which did not exhibit suppression on lymphocyte mitogenesis, Yam 1B SN had several characteristics that differ from TGF-l3. According to Kehrl et al., I2213TGF-B inhibits the proliferation and immunoglobulin secretion of human B cells, but not immunoglobulin production by an EBV-transformed B-cell line, CESS cells. Though we did not examine it by using CESS cells, Yam 1B SN suppressed immunoglobulin generation by EBV-transformed B-cell lines, Yam 3B1, and HLN-Ith cells (data not shown). The inhibitory activity of proliferative response induced by TGF-B was partly overcome by the addition of IL-2,12 but that induced by Yam 1B SN was not (data not shown). It has been reported that mitogen-activated human B lymphocytes release a very small quantity of TGF-13,12and that the biological activity by TGF-B on B lymphocytes is abrogated by the addition of an antibody specific for TGF-B,.13 Among TGF-B family, TGF-P,, TGF-B,, and TGF-B, are well studied.22 TGFpl, TGF-B,, and TGF-B, are equipotent in the degree to which they inhibit cell growth. TGF-B, is functionally similar to TGF-B, in its inhibition of B-cell proliferation.13 The polyclonal antibody for TGF-j3, shows the similar effect of neutralization of biological activities of both TGF-B, and TGF-j3,. The inhibitory activity of the Yam 1B factor was not abrogated after being neutralized by this anti-TGF-B antibody (Table 3). TGF-B is secreted by culture cells in an inactive or latent form in general. Several physicochemical treatments with acidification (less than pH 4), alkalinization (greater than pH 9),” or proteinases” will activate latent TGF-B. Proteolytic cleavage of TGF-B by the serine proteinase plasmin is relevant to physiological mechanism of activation.25 In contrast, Yam 1B inhibitory factor secreted by Yam 1B cells showed an activity without any treatment. Physicochemically, the activity of Yam 1B SN was abrogated by alkalinization (pH 10.0) or by pronase treatment. Activity of Yam 1B SN was not intensified with acidifacation but was slightly weakened. Crude TGF-l3 materials have been applied to a cation-exchange chromatography for purifica-
factor
produced
by a B-cell
line
/ 615
tionZ6 but the Yam 1B SN was not adsorbed in cation-exchange chromatography (data not shown). These results suggested that the inhibitory activity in the Yam 1B SN appeared to be unrelated to TGF-B activity. However, whether Yam 1B SN contains a new factor different from TGF-B remains unknown until the sequence of Yam 1B factor is obtained. Further purification and characterization of the inhibitory factors derived from Yam 1B is now under progress. Several other substances that inhibit lymphocyte function have been identified. For example, soluble immune suppressor supernatant (SISS)-B is produced by ConA-activated T lymphocytes. SISS-B has an MW, of 60,000 to 80,000, is stable at pH 2.5, and labile at 56°C although it has not been determined whether SISS-B inhibits B-cell proliferation. Soluble immune response suppressor (SIRS) and monoclonal nonspecific suppressor factor (MNSF) are murine suppressor factors that inhibit antibody secretion by B cells. These factors differ in MW, and sensitivity to heat and pH because they have a smaller MW, (14,000 to 24,000) than the suppressor fractions described here, are stable at 56°C and unstable at pH 2.0.5,6 A recent study by Lau et a1.27 shows that a suppressor-activating factor (SAF) produced by a mutant T-cell line is found to reside in a oxidized product of spermidine (SDA). Yam IB SN-derived factor appeared inconsistent with it being SDA because Yam 1B SN was prepared in serum-free medium after 3 days of culture. Yam IB SN is very sensitive to both heat and pronase treatment, which indicates that Yam IB SN is a protein. It has been reported that an EBV-transformed B-cell line secretes B-cell growth factors (BCGF) that sustain the proliferation in an autocrine 10op.~~Thus, it seems reasonable to assume that a similar autocrine inhibitory mechanism may function in cellular homeostasis as previously reported.” The putative Yam 1B factor may be one of a family of such autocrine growth inhibitory factors. It is interesting that Yam 1B cell line was derived from an ATL patient. It is well-known that ATL-T cells secrete several soluble growth factors; however, reports about B cells derived from ATL are very rare. It is known that the preferred target cells for HTLV-1, an etiologic agent of ATL, are CD4-expressing T cells, but B cells can be also infected with HTLV-1.29”3 The infected B cells are shown to have ATLA as well as EBNA29.32,33and IL-2R antigen (C~25).?9,30,32.33 Therefore, Yam 1B did not seem to be infected with HTLV-1. However, recent investigation showed that some infected B-cell lines have HTLV-1 clonally integrated without production of viral antigens.30,3’The integrated proviral DNA in the Yam 1B cell line is currently under investigation.
616 I Morikawa et al.
CYTOKINE,
MATERIALS AND METHODS Cell Lines Human B-lymphoblastoid cell line Yam 1B was originally derived from the peripheral blood of an adult T-cell leukemia (ATL) patient. The cell line has been established in our laboratory and has been maintained in RPM1 1640 supplemented with 10% fetal calf serum (FCS; Flow Laboratories, Mclean, VA) since 1985. The Yam 1B cell line showed common B-cell markers, including cell surface immunoglobulin (SIg), CD23, HLA-DR, and was negative for T cell (CD2, 3,4,8), myelomonocytic (CD13, 16), and CD25 (IL-2R, ~55) cell surface antigens. The cells possessed EBV-associated antigen (EBNA), but not adult T-cell-associated antigen (ATLA). In addition, the Yam 1B cell line had cytoplasmic IgM and spontaneously secreted small amounts of IgM (10 to 100 ng/mL) in the culture supernatants. EBV-transformed human B-cell lines, HLN-Ith and Yam 3B1, 3B0, and a pre-T-cell line, HPB-MLp-W, were also established in our laboratory and have been maintained in continuous culture for approximately 2 years. Other cell lines, Daudi, ARH 77, MOLT-4, and K 562, have been maintained in our laboratory for several years. All cell lines used in this experiment were mycoplasma-free on repeated testing.
Preparation
of Inhibitory
Factor Supernatants
For generation of inhibitory factor in serum-free medium, Yam 1B cells were washed and resuspended at 5 x 105/mL in serum-free RPM1 1640 medium at 37°C in a 5% CO, atmosphere. After 72 h of culture, cell-free supernatant fluids were harvested by centrifugation and were stored at -20°C until used. Serum-free Yam 1B SN were subsequently concentrated 50-700-fold for either assayof crude inhibitory factor activity or gel filtration under pressure using a ultrafiltration system with a PM 10 filter and with Centriprep-10 (Amicon Corp., Danvers, MA).
Gel Filtration Six milliliters of 700-fold concentrated Yam 1B SN was applied to a column (1.2 x 107 cm) of Sephacryl S-300 (Pharmacia), equilibrated with 20 mM Tris-HCL buffer containing 0.3 M NaCL (pH 7.2) at 4°C. Each fraction (2.3 mL) was filtered through 0.45 km membrane before being tested at a 1:4 dilution for effects on the cell proliferation. The inhibitory factor-containing fractions were collected, ultrafiltrated, and refractionated on the same columns. Rechromatographed fractions were retested for the inhibitory activity. The column was calibrated with cytochrome c(MW, 13,000), ovalbumin (MW, 43,000), bovine serum albumin (BSA: MW, 67,000), lactic dehydrogenese (LDH; MWr 140.000), and ferritin (MW, 440,000).
Anion-Exchange
Chromatography
The active fractions eluted from Sephacryl S-300 column were concentrated by ultrafiltration using Centriprep 10 and dialyzed extensively against 20 mM Tris-HCL buffer (pH 8.0) and applied to a DEAE-Cellulofine A-500 (Seikagaku Ko-
Vol. 3, No. 6 (November
1991: 609-618)
gyo) column equilibrated with the same buffer. After the column was washed with the starting buffer, the material was eluted with a linear gradient of 20 mM Tris-HCL buffer containing 0 to 0.5 M NaCL (PH. 8.0).
Protein Determination Protein concentration was determined either by using the Bio-Rad Laboratories protein assay or by measuring absorbance at 280 nm. Bovine serum albumin was used as a standard.
Cell Preparation Human tonsils were obtained by tonsillectomy from juvenile patients with chronic tonsillitis and were dispersed into single cell suspensions. Mononuclear cells were isolated on a Ficoll-Hypaque gradient. PBMCs were obtained from healthy donors. Monocytes and natural killer cells were depleted by incubation with 5 mM L-leucine methyl ester (Sigma, St. Louis, MO) in serum-free medium.34 T cellswere separated by rosetting twice with 2-aminoethylisothiouronium-bromide (Sigma)-treated sheep erythrocytes (E) and were obtained from rosetted (E+) cells by treating the attached E with lysing buffer [NH,CL:KHCO,] and then passing them over a nylon wool column. Nonrosetted (E-) cells were additionally purified by isolating small dense B cells from 60%/70% interface of a discontinuous percoll gradient (Pharmacia).
Assay for Inhibitory
Effect on Cell Proliferation
To test for inhibitory activity on the cell proliferation, 1 x lo5 cells were incubated with reciprocal dilutions of the concentrated Yam 1B SN preparations or control preparations and appropriate mitogens in RPM1 1640 supplemented with 10% FCS, L-glutamin, 2 mercaptoethanol, and penicillinstreptomycin. Cultures were stimulated with PHA-P; 1:lOO dilution (Difco Laboratories, Detroit, MI), ConA; 10 ng/mL (Pharmacia), phorbol myristate acetate (PMA); 10 ng/mL (Sigma), or SAC; 1:105 vol/vol (Calbiochem, Behring Co., CA). The cultures were incubated for 72 h in a 5% CO, incubator and pulse-labeled with 1 yCi [3H]thymidine for the final 18 h of culture. The cells were collected onto glassfilter paper using an automated cell harvester and the incorporated radioactivity was assessedby liquid scintillation counting. In some experiments, the effect of inhibitory factors on the proliferation of lymphoblastoid cell lines was determined by culturing 1 X lo5 cells in the presence of Yam 1B SN preparations or control preparations.
Assay for the Inhibitory Generation
Effect on Immunoglobulin
In assessingthe effect of Yam 1B SN on cell differentiation, PBMC was cultured at 1 x lo5 cellsin 200 PL containing RPM1 1640 with 10% FCS and varying concentrations of Yam 1B SN or control medium in the presence or absence of PWM (1:50 dilution; Gibco, Grand Island, NY) for 7 days. After that, the culture supernatants were harvested and the amount of immunoglobulin in the culture supernatants was determined by enzyme-linked immunosorbent assay(ELISA).
Immunoregulatory
factor produced by a B-cell line / 617
In some experiments, EBV-transformed human B-cell lines, which spontaneously secrete IgM and IgG (Yam 3Bl) and IgA (HLN-Ith), were used as target cells for evaluating the inhibitory effect of Yam 1B SN. The cell lines were cultured at 5 x lo4 cells per well in the presence or absence of Yam 1B SN for 4 days.
incubation at 4°C or -20°C for 48 h; heating at 56” or 80°C for 10 min; incubation at 37°C with and without insoluble protease (100 kg/mL; Sigma) or insoluble trypsin (100 kg/mL; Sigma) for 2 h; acidified to pH 2.0 with HCL or alkalized to pH 10.0 with NaOH for 6 h followed by adjustment to pH 7.2 and to the original volume.
Enzyme-Linked
Cytokines and Antibodies
Immunosorbent
Assay
Total immunoglobulin was determined by using ELISA as described in detail.3s In brief, 100 FL of the culture supernatants was added to microculture plates pre-coated with affinity-purified goat anti-human immunoglobulin (Cappel Laboratories, West Chester, PA). After incubation overnight at 4°C the microplates were washed and mixed with alkaline-phosphatase-conjugated anti-human IgM, IgG, and IgA (Cappel). After 2 h incubation at 37°C the plates were washed and the substrate paranitrophenylphosphate (Sigma) was added to each well. Absorbance at 405 nm was measured with Easy Reader EAR400 (SLT-Labinstruments, Austria). In each experiment, a standard curve was constructed from the samples containing a known concentration of immunoglobulin from normal human serum.
Immunojfuorescence For the detection of cell surface activation antigens, the cells were incubated with OKT9, a monoclonal antibody (MoAb) specific for transferrin R (Ortho Diagnostic Systems, Tokyo, Japan) or Tat-1 (anti-IL-2 R MoAb, CD25: donated by Dr. T. Uchiyama) for 20 min on ice, washed twice, and incubated with fluorescein isothiocyanate (FITC)labeled goat antibody to mouse IgG, F (ab’), (Cappel). Cell surface immunofluorescence was analyzed with FACScan (Becton-Dickinson, Mountain View, CA).
Phytohemagglutinin-Induced Production of Interleukin 2 in Human Peripheral Blood Lymphocytes Human PBMC was cultured at 1 X lo6 cells/mL (1 mL/well) in 24-well plates with or without Yam 1B SN in the presence or absence of PHA-P (1500 dilution) for 48 h at 37°C in a 5% CO, incubator. The culture medium consisted of RPM1 1640 with 1% FCS. The supernatants were recovered by centrifugation and were stored at -20°C until assay.
Assay of Interleukin
2 Activity
CTLL cells were washed extensively and were resuspended at 1 x lo5 cells/mL in the culture medium. One hundred microliters of the suspension was seeded in each well of flat-bottomed microtiter plate wells with 100 p,L of reciplocal dilution of sample supernatants. After 18 h of culture, 1 PC1 of [3H]thymidine was added to each well for an additional 6 h. The incorporated radioactivity was determined as described above for blastogenic response of PBMC.
Examination Supernatant Fifty-fold free condition
of Physicochemical
Nature of Yam 1B
concentrated Yam 1B SN produced in serumwas subjected to the following treatment:
Human recombinant and natural TGF-l3 were donated by Dr. T. Ikeda (King Brewing Co., Kakogawa, Japan). TGF+ neutralizing antibody was obtained from R & D Systems (Minneapolis, MN). Anti-TGF-l3 antibody was prepared from highly specific IgG fraction of rabbits immunized with highly purified, native porcine platelet TGF-l3. The antibody has been found to have a high titer for neutralization of the biological activities of TGF-l3, and TGF-6,. The antibody does not show any cross-reactivity with acidic or basic fibroblast growth factors, platelet-derived growth factor, or epidermal growth factor. Human IL-2 was purchased from Boehringer (Mannheim, Germany). IL-2 was produced by stimulating normal human T cells with PHA, and the culture supernatants were purified by using a sequence of ion-exchange chromatography and gel filtration. The resulting preparation was free of interferon and mitogenic concentrations of PHA (PHA concentration < 1 ng/mL).
Antibody Treatment The inhibitory factor-containing culture SN was preincubated with a previously determined optimal concentration of a rabbit antibody that neutralizes 1 p,g/mL of TGF-l3 (50 kg/mL) for 2 h at 4°C. Equivalent concentrations of normal rabbit IgG (Cappels) were added to parallel aliquots of SN as a control.
Transforming
Growth Factor p Assays
Supernatants to be assayed for TGF-l3 were obtained from partially purified Yam 1B SN by Sephacryl S-300 gel filtration. The supernatant was dialyzed with 0.2 M acetic acid before being assayed for TGF-l3. The bioassay for TGF-l3 depends on the growth inhibition of Mv 1 Lu mink lung epithelial cells (Ccl-64; American Type Culture Collection) measured by the [‘251]IdU incorporation into the DNA of actively growing cells after 72-h culture at 37°C in 5% CO, as previously described.36
Acknowledgment
The authors thank Dr. Tatsuhiko Ikeda for TGF-P assay and Akiko Kawakami for preparation of the manuscript. REFERENCES 1. Fleisher TA, Greene WC, Blaese RM, Waldmann TA (1981) Soluble suppressor supernatants elaborated by concanavarin A-activated human mononuclear cells. II. Characterization of a soluble suppressor of B cell immunoglobulin production. J Immunol 126:1192-1197.
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Proteolytic fibroblast-
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