TGF-β has marked inhibitory effects on the immune system but also serves as a costimulatory factor in the development of T cells with down-regulatory activities. This cytokine is secreted as a latent complex and converted extracellularly to its active form. We have recently learned that anti-CD2 is a potent inducer of lymphocyte-derived TGF-β and that NK cells are the predominant source. The objective of this study was to compare levels of constitutive, anti-CD2-induced and cytokine-regulated TGF-β produced by blood lymphocytes from patients with systemic lupus erythematosus (SLE) in comparison with healthy controls. Using a highly sensitive and specific bioassay to assess TGF-β, we report that unstimulated PBL from SLE patients, especially the NK cell subset, produced decreased levels of active TGF-β. In response to anti-CD2, concentrations of active and total TGF-β were also decreased in SLE. After learning that IL-2 and TNF-α enhance lymphocyte production of active TGF-β, we found that the addition of these cytokines was unable to increase active TGF-β to normal concentrations. Although we observed that IL-10 inhibited the production of active TGF-β, antagonism of this cytokine was unable to completely correct the defect. In two SLE patients with B cell hyperactivity, spontaneous IgG production was almost abolished by the combination of TGF-β and IL-2. Therefore, decreased production of each of these cytokines in SLE could be important in the perpetuation of B cell hyperactivity.

Systemic lupus erythematosus (SLE)3 is a disorder of generalized autoimmunity characterized by B cell hyperactivity with numerous autoantibodies against nuclear, cytoplasmic, and cell surface Ags. This autoimmune disease has a multifactorial pathogenesis with genetic and environmental precipitating factors (1). SLE is a T cell-dependent disease with many examples of T cell dysfunction. Mechanisms that maintain self tolerance and down-regulate B cell function have broken down (reviewed in 2 . Since spontaneous remissions are frequent in human SLE (3), dysfunctional regulatory cells may contribute to perpetuation as well as the onset of SLE.

Recently, studies from this laboratory have revealed that TGF-β is an important costimulatory factor in the generation of CD8+ T cells that down-regulate B cell function (4). TGF-β is a multifunctional family of cytokines important in tissue repair, inflammation, and immunoregulation (5). Lymphocytes and monocytes produce the β1 isoform of this cytokine (6). TGF-β is different from most other cytokines in that it is secreted as an inert precursor molecule and converted to its biologically active form extracellularly (5, 7). Although monocytes have been considered to be the principal source of this cytokine in human peripheral blood (8, 9), we report (10) that NK cells are also a major source of TGF-β and can convert the latent complex to its active form. In addition, we have demonstrated that the combination of IL-2 and TGF-β can condition activated CD8+ T cells to develop suppressor effector function (10).

Because of the apparent importance of TGF-β in regulatory cell development and our previous observation of dysfunctional CD8+ T cells in SLE (11), we decided to quantify the production of this cytokine. Since active TGF-β comprises only a small fraction of the total amount, a sensitive and specific bioassay was needed for this measurement. The genetically engineered mink cell line generated by Rifkin et al. provided such an assay (12).

Previously, we reported that anti-CD2 mAb can stimulate T cells to proliferate in the absence of monocytes. Unlike anti-CD3, resting B cells can serve as accessory cells for similar stimulation with anti-CD2 (13). In a companion study in this issue, we demonstrate that unlike anti-CD3, anti-CD2 is a potent inducer of active TGF-β (10). To broaden our capacity to induce TGF-β production, we describe herein that IL-2 and TNF-α have this property, whereas IL-10 has the opposite effect. These findings are particularly relevant to SLE, since production of IL-2 and TNF-α production is decreased (14, 15, 16) and IL-10 production is increased (17, 18). We report decreased production of lymphocyte-derived TGF-β in SLE, which cannot be normalized by the addition of recombinant IL-2 and TNF-α or by antagonism of IL-10. To determine the possible clinical relevance of cytokine production defects in SLE, we have initiated studies to determine whether B cell hyperactivity in SLE can be down-regulated by IL-2 and TGF-β ex vivo and have obtained preliminary evidence to support this possibility.

Thirty-eight subjects with a diagnosis of SLE that fulfilled at least four of the 1982 revised criteria for the classification of SLE (19) were studied. The group consisted of 35 women and 3 men (33 Hispanic, 3 Asian, 2 African American). The mean age was 34.3 years (range, 20–75 years). Nineteen patients were hospitalized, and 19 were attending the outpatient clinic. Most of the hospitalized patients were untreated before admission and were studied before they received corticosteroids. Outpatients were receiving <20 mg of prednisone, and none were receiving cytotoxic drugs. Disease activity was assessed with the SLAM (20) and SLEDAI (21) indices with mean values of 9.2 and 10.2, respectively. Healthy donors served as controls and were matched as closely as possible for age, sex, and ethnic group.

Antibodies used were anti-CD2 (OKT11, American Type Culture Collection (ATCC), Rockville, MD, and GT2 made available by Dr. Alain Bernard, Nice, France) (22); anti-CD3 (454, a gift from Dr. William Stohl, Los Angeles, CA) (23); anti-CD74 (L243, ATCC, MD); rTGF-β1 and anti-TGF-β (1D11.16), a murine IgG1, were kindly provided by Dr. Bruce Pratt (Genzyme Pharmaceuticals, Farmington, MA) (24). Anti-CD16 (3G8) was provided by Dr. Jay Unkeless, New York, NY. TNF-α and IFN-γ were purchased from R&D Systems, Minneapolis, MN. IL-2 was purchased from Cetus, Emmeryville, CA. IL-10 was kind gift from Satwant Narula (Schering Plough Pharmaceuticals, Kenilworth, NJ), as was anti-IL-10 (JES3–19F1) (25) and control rat IgG2a.

PBMC were prepared from heparinized venous blood by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density gradient centrifugation. The mononuclear cells were washed in PBS with 5 mM EDTA (Life Technologies, Grand Island, NY) to remove platelets, which are a rich source of TGF-β (26). Lymphocytes and monocytes were separated from PBMC by centrifugation through a continuous Percoll (Pharmacia) density gradient (27). The percentage of monocytes remaining in the high density, lymphocyte-enriched fraction was somewhat higher in SLE (8.5% vs 4.3%). The percentage of lymphocytes remaining in the monocyte-enriched low density cells was similar in SLE and controls (20.4 vs 21.3%).

NK and T cells were prepared as described previously (4). PBL were immediately rosetted with 2-aminoethylisothiouronium bromide-treated SRBC (28). The nonrosetting cells were then incubated with anti-CD3 and anti-CD74 (anti-HLA-DR) Abs on ice and depleted of reacting cells using immunomagnetic beads (Dynal, Great Neck, NY). The percentage of CD56+ cells in this fraction was similar in SLE and controls (84.0 vs 83.2%). T cells were prepared from rosetting cells by negative selection following depletion of CD16+ and CD74+ cells also using immunomagnetic beads (Dynal). The percentage of CD3+ cells in this fraction was usually >95%.

Procedures for cell cultures have been described previously (4). In brief, 1 × 105 of the various mononuclear cell populations were added to the wells of 96-well flat-bottom microtiter plate (Greiner Rocky Mountain Scientific, Salt Lake City, UT). These plates were selected following a comparison of the nonspecific binding of TGF-β to commonly used tissue culture plates. The lymphocytes were suspended in AIM V serum-free medium (Life Technologies) since serum contains significant amounts of latent TGF-β. Some lymphocytes were stimulated with the optimal concentrations of anti-CD2 to induce TGF-β production (GT2 1:40 and T11 1:80) hybridoma culture supernatants.

Mink lung epithelial cells (MLEC) that had been transfected with an expression construct containing a plasminogen activator inhibitor (PAI-1) promoter fused to luciferase reporter gene were kindly provided by D. B. Rifkin, New York, NY. The procedure described by this group was used to assay TGF-β (28). MLEC (2 × 104/well) were incubated with 200 μl of supernatant for 18 h at 37°C. To assay for luciferase activity, MLEC were lysed by a cell lysis reagent (Analytical Luminescence, Ann Arbor, MI). Cell lysates were then reacted with assay buffer and luciferin solution (both from Analytical Luminescence) immediately before being measured in a luminometer (Lumat, Berthold Analytical Instruments Inc., Nashua, NH). To measure total TGF-β activity, the samples were heated at 80°C for 3 min to release the active cytokine from the latent complex. To measure active TGF-β activity, the supernatants were examined without heating. In all assays, several concentrations of rTGF-β were included to generate a standard curve. The variability of triplicate cultures was <10% of the mean value.

PBMC (2 × 105) from patients with active SLE were cultured in serum-free (AIM V) culture medium in the wells of a 96-well flat-bottom microtiter plate. For the first 3 days, the PBMC were cultured at 37°C in 5% CO2 in a humidified incubator with or without IL-2 (10 U/ml) and/or TGF-β (10 pg/ml). The medium was then removed, and after the cells were washed, fresh serum-free medium was added, and the cells were cultured for 7 more days. The supernatants were harvested and assayed for IgG content by an ELISA, as described previously (29).

The significance of the results was analyzed by Student’s t test or the Mann-Whitney test performed using GBSTAT software (Professional Statistics and Graphics Computer Program, Dynamic Microsystems Inc., Silver Spring, MD).

To verify the specificity of the TGF-β bioassay used, we added a neutralizing anti-TGF-β mAb to the culture supernatants of unstimulated and anti-CD2-stimulated PBL. In the four experiments shown in Table I, this procedure abolished >98% of the mink cell response.

Table I.

Specificity of the TGF-β assaya

Expt.Anti-CD2Medium (pg/ml)Anti-TGF-β (pg/ml)mIgG1
− 229 <5 279 
 593 <5 284 
− 210 <5 360 
 445 <5 288 
− 722 <5 764 
 1365 <5 1359 
− 3807 <5 2121 
 >8000 96 3886 
Expt.Anti-CD2Medium (pg/ml)Anti-TGF-β (pg/ml)mIgG1
− 229 <5 279 
 593 <5 284 
− 210 <5 360 
 445 <5 288 
− 722 <5 764 
 1365 <5 1359 
− 3807 <5 2121 
 >8000 96 3886 
a

PBL (1 × 105 were incubated with or without anti-CD2 for 48 h, and the supernatants were harvested. Anti-TGF-β (10 μg/ml) or an equivalent amount of control mouse IgG1 was added, and the supernatant was examined for total TGF-β activity using mink cells (MLEC) with a luciferase construct.

We first compared lymphocytes from control donors and SLE patients for their ability to produce total TGF-β either constitutively or after stimulation with anti-CD2 (Fig. 1 A). Constitutive production of total TGF-β by control PBL was somewhat more than that of patient lymphocytes (623 ± 107 vs 385 ± 98 pg/ml). The response to anti-CD2 by control PBL was significantly more vigorous than SLE PBL (1289 ± 224 vs 586 ± 99, p = 0.03). NK cells were the principal source of this cytokine. During this 48-h culture period, production of total TGF-β by T cells was barely detectable. Although we had an insufficient amount of blood from SLE patients to prepare B cells, the amount of total TGF-β produced by this lymphocyte subset from healthy donors was generally <200 pg/ml (see Ref. 10, Table IV).

FIGURE 1.

Production of TGF-β by unstimulated and anti-CD2-stimulated human peripheral blood lymphocyte populations. A, Various lymphocyte populations from healthy donors and SLE patients were added to microtiter plates at 1 × 105/well. Some wells received the anti-CD2 mAbs GT2 (1:40) and T11 (1:80). After 2 days at 37°C, the supernatants were harvested and assayed for total TGF-β by heating the samples at 80°C for 3 min. B, Active TGF-β was measured without heating the supernatants. Significant p values as determined by the t test are indicated.

FIGURE 1.

Production of TGF-β by unstimulated and anti-CD2-stimulated human peripheral blood lymphocyte populations. A, Various lymphocyte populations from healthy donors and SLE patients were added to microtiter plates at 1 × 105/well. Some wells received the anti-CD2 mAbs GT2 (1:40) and T11 (1:80). After 2 days at 37°C, the supernatants were harvested and assayed for total TGF-β by heating the samples at 80°C for 3 min. B, Active TGF-β was measured without heating the supernatants. Significant p values as determined by the t test are indicated.

Close modal

Although most constitutive, lymphocyte-derived TGF-β is in the latent form, the biologically active form was also detectable. The amount of active TGF-β from control donors’ and SLE patients’ lymphocytes is shown in Fig. 1 B. Similar to levels of the latent complex, active TGF-β detected in culture supernatants from controls was approximately twice as much as that in SLE patients. As with total TGF-β, NK cells were the principal source of active TGF-β, and control NK cells produced significantly more than those from SLE patients (98 ± 37 vs 39 ± 13 pg/ml, p = 0.01). Active TGF-β from T cells was not detectable, and that from control B cells was only barely detectable (results not shown; see Ref. 10, Table V).

The response to stimulation with anti-CD2 was significantly greater in controls (p < 0.001). Again NK cells appeared to be the predominant source of this cytokine. However, since the relative increase by NK cells was less than that of PBL, other lymphocyte populations might contribute to the extracellular conversion of latent to active TGF-β. Production of this cytokine by T cells was negligible during this interval. Thus, defects in both total and active TGF-β were documented in SLE that could be largely attributed to NK cells.

Since monocytes are a major source of TGF-β, we compared the production of the total and active form in SLE and controls (8, 9). As shown in Table II, there were no significant differences in the two groups.

Table II.

Monocyte TGF-β production in SLE and healthy controlsa

TGF-β (pg/ml)
TotalActive
Controls (14) 817 ± 295 68 ± 23 
SLE (20) 674 ± 205 54 ± 15 
TGF-β (pg/ml)
TotalActive
Controls (14) 817 ± 295 68 ± 23 
SLE (20) 674 ± 205 54 ± 15 
a

Monocytes (1 × 105) were cultured for 48 h, and the supernatants were tested for TGF-β. Numbers in parentheses indicate the number of subjects studied.

We next investigated the kinetics of lymphocyte TGF-β production by measuring TGF-β activity over several days. The results in Figure 2 show that SLE production of TGF-β was always lower than that of control donors. Most striking was the difference in the production of total and active TGF-β following stimulation with anti-CD2, a difference that increases with time.

FIGURE 2.

Time course of TGF-β production. PBL from 12 SLE patients and 12 healthy controls were added to microtiter plates (1 × 105/well) as described and cultured with or without anti-CD2 for 1 to 4 days at 37°C. The harvested supernatants were assayed for both total and active TGF-β by the MLEC assay.

FIGURE 2.

Time course of TGF-β production. PBL from 12 SLE patients and 12 healthy controls were added to microtiter plates (1 × 105/well) as described and cultured with or without anti-CD2 for 1 to 4 days at 37°C. The harvested supernatants were assayed for both total and active TGF-β by the MLEC assay.

Close modal

It has been established that blood mononuclear cell production of IL-2, TNF-α, and IFN-γ is decreased in SLE and that IL-10 production is increased (14, 15, 16, 17, 18). We selected, accordingly, these four cytokines for study. PBL from healthy controls were incubated for 48 h with doses over a 3-log concentration range. Figure 3 shows that IL-2 and TNF-α increased the production of active TGF-β in a dose-dependent manner. IFN-γ, however, had no effect, and IL-10 was suppressive.

FIGURE 3.

Effects of various cytokines on lymphocyte production of active TGF-β. PBL (1 × 105/well) were cultured with IL-2 (1, 10, and 100 U/ml), TNF-α (0.1, 1, and 10 ng/ml), IFN-γ (1, 10, 100 U/ml), and IL-10 (0.1, 1, and 10 ng/ml) for 2 days.

FIGURE 3.

Effects of various cytokines on lymphocyte production of active TGF-β. PBL (1 × 105/well) were cultured with IL-2 (1, 10, and 100 U/ml), TNF-α (0.1, 1, and 10 ng/ml), IFN-γ (1, 10, 100 U/ml), and IL-10 (0.1, 1, and 10 ng/ml) for 2 days.

Close modal

On the basis of these results, we selected five SLE patients with active disease who previously demonstrated decreased TGF-β production in response to anti-CD2. As before, the response to anti-CD2 in SLE was markedly less than that by controls (Table III). In this small group, constitutive production of active TGF-β was significantly decreased in SLE. The addition of IL-2 and TNF-α proportionally increased levels of active TGF-β in both groups but was unable to correct the defect.

Table III.

IL-2 and TNF-α could not correct the defect in active TGF-β production in SLEa

TGF-β (pg/ml)
Healthy donors (n = 5)SLE (n = 5)p
Nil 41 ± 12 9 ± 2 0.032 
Anti-CD2 290 ± 81 31 ± 7 0.013 
IL-2 (10 U/ml) 69 ± 7 13 ± 4 <0.001 
TNF-α (10 ng/ml) 77 ± 19 22 ± 8 0.037 
IL-2 and TNF-α 131 ± 21 38 ± 14 0.006 
TGF-β (pg/ml)
Healthy donors (n = 5)SLE (n = 5)p
Nil 41 ± 12 9 ± 2 0.032 
Anti-CD2 290 ± 81 31 ± 7 0.013 
IL-2 (10 U/ml) 69 ± 7 13 ± 4 <0.001 
TNF-α (10 ng/ml) 77 ± 19 22 ± 8 0.037 
IL-2 and TNF-α 131 ± 21 38 ± 14 0.006 
a

PBL (1 × 105) were cultured for 48 h with the above additives, and the supernatants were tested for active TGF-β.

Since IL-10 decreased levels of active TGF-β and production of this cytokine is increased in SLE (17, 18), we investigated the effect of antagonizing spontaneously produced IL-10 with a neutralizing mAb. In this subgroup of SLE patients, constitutive active TGF-β was again significantly decreased (Fig. 4). The addition of anti-IL-10 increased active TGF-β in both SLE and controls, and this effect was relatively greater in SLE so that the differences between these two groups were no longer significant. Following anti-CD2 stimulation of PBL, anti-IL-10 had no effect on TGF-β activity. Since monocytes are the principal source of IL-10, PBMC were cultured in parallel with PBL in three experiments with similar results (not shown).

FIGURE 4.

Effects of anti-IL-10 Ab on constitutive and anti-CD2-stimulated active TGF-β production. PBL from 16 SLE patients and 11 healthy controls (1 × 105/well) were cultured with or without anti-IL-10 or control rat IgG2a (10 μg/ml) for 2 days. Values for constitutive and anti-CD2 stimulated active TGF-β are shown. Relevant p values are indicated (Mann-Whitney).

FIGURE 4.

Effects of anti-IL-10 Ab on constitutive and anti-CD2-stimulated active TGF-β production. PBL from 16 SLE patients and 11 healthy controls (1 × 105/well) were cultured with or without anti-IL-10 or control rat IgG2a (10 μg/ml) for 2 days. Values for constitutive and anti-CD2 stimulated active TGF-β are shown. Relevant p values are indicated (Mann-Whitney).

Close modal

In view of recent evidence that TGF-β is an important costimulatory factor in the development of T suppressor cells (4), the decreased amounts of active TGF-β in SLE we have documented might contribute to the inability of CD8+ T cells to down-regulate B cell activity. We have recently observed that a brief exposure of CD8+ T cells from healthy individuals to TGF-β and IL-2 enables them to down-regulate Ig production (Figure 6 in 10 . We considered that similar exposure of SLE lymphocytes to these cytokines might condition them to down-regulate spontaneous IgG production. Examples of this cytokine-mediated suppression of Ig production are shown in Figure 5. PBMC from two SLE patients were exposed to IL-2 (10 U/ml), TGF-β (10 pg/ml), or both of these cytokines for 72 h and subsequently cultured for an additional 7 days. In case 1, neither IL-2 nor TGF-β alone had significant effects, whereas in case 2 each of these cytokines appeared to have some effect. In both cases, a brief exposure of PBMC to both IL-2 and TGF-β resulted in the suppression of spontaneous IgG production by 85%. The mechanism responsible for this effect is the subject of current investigation.

FIGURE 5.

The effects of TGF-β and/or IL-2 on spontaneous IgG production by PBMC from patients with SLE. PBMC (2 × 105/well) were cultured with or without IL-2 (10 U/ml) or TGF-β (10 pg/ml) for 3 days. The cells were washed, and the cells were cultured in fresh medium for an additional 7 days. The harvested supernatants were assayed for IgG by an ELISA.

FIGURE 5.

The effects of TGF-β and/or IL-2 on spontaneous IgG production by PBMC from patients with SLE. PBMC (2 × 105/well) were cultured with or without IL-2 (10 U/ml) or TGF-β (10 pg/ml) for 3 days. The cells were washed, and the cells were cultured in fresh medium for an additional 7 days. The harvested supernatants were assayed for IgG by an ELISA.

Close modal

This is the first report, to our knowledge, documenting the capacity of circulating blood lymphocytes from patients with an autoimmune disease to produce active TGF-β. Levels of both total and active TGF-β in short term culture supernatants of unstimulated and stimulated lymphocytes from SLE patients were decreased in comparison with normal controls. Previously, a relative decrease in active TGF-β produced by T cell lines from patients with active multiple sclerosis had been reported (30). Although the decreased levels of TGF-β activity in SLE most likely reflect decreased production, these differences could also be due to increased consumption by TGF-β receptors. Further studies will be needed to exclude this possibility.

In an accompanying report, we have found that NK cells are the principal source of both total and active TGF-β produced by unstimulated lymphocytes (10). In SLE and healthy controls, NK cells produced substantially more TGF-β than T cells (Fig. 1). Decreased amounts, however, of both constitutive and induced NK cell-derived TGF-β were found in SLE, and these defects were found in amounts of the total and active form of this cytokine. Thus, in addition to the well-known defect of NK cell cytotoxic activity in SLE (31, 32), production of TGF-β appears to be decreased as well.

While resting human T cells produced trivial amounts of TGF-β during the first 48 h of culture, stimulated T cells can produce significant quantities of this cytokine (33). Although many T cells appear to be chronically stimulated in SLE (34, 35), they produced only minimal amounts of TGF-β.

Other sources of TGF-β include B cells and monocytes. Although we also reported that resting B cells from healthy donors produce small amounts of TGF-β (10), these lymphocytes have the capacity to produce this cytokine (36, 37). Moreover, IgG secreted by Ag-activated B cells has been found to be complexed latent TGF-β (38). When this complex is bound to FcR on macrophages, latent TGF-β is converted to its active form and may be immunosuppressive (39). Monocytes have been considered to be the principal hemopoietic source of TGF-β (8, 9). Differences in monocyte-derived total and active TGF-β between SLE patients and controls were not found (Table II).

It is important to emphasize that this report concerns short term production of TGF-β. We have focused our attention on this interval because CD8+ T cells require the presence of picogram per milliliter quantities of active TGF-β coincident with activation for development of down-regulatory function (10).

Although stimulated T cells do not produce much TGF-β during the initial 72 h, they can produce considerable quantities of latent TGF-β at later periods (33; K. Ohtsuka and D. A. Horwitz, unpublished observations).

Decreased concentrations of TGF-β in SLE did not appear to correlate with disease activity. To date, TGF-β production has been measured in a group of 10 subjects with rheumatoid arthritis, and a modest decrease of anti-CD2-induced total and active TGF-β has been observed. Larger numbers of RA patients are required to determine the significance of this finding.

Unlike anti-CD3, anti-CD2 mAbs strongly stimulate lymphocytes to produce total and active TGF-β (10). Following stimulation with anti-CD2, both production of total and active TGF-β was markedly reduced in SLE. Because the lymphocyte mitogenic response to anti-CD2 in SLE is also reduced (40, 41), decreased TGF-β production might reflect decreased signaling through the CD2 pathway rather than a decreased capacity of SLE lymphocytes to produce TGF-β. We, therefore, turned our attention to cytokine regulation of TGF-β production.

Concentrating on cytokines known to be abnormally produced in SLE, we have shown that both TNF-α and IL-2 increase lymphocyte-derived active TGF-β and that IL-10 has the opposite effect. To our knowledge, these findings have not been previously described. Using other cellular targets, IL-2 has been reported to increase TGF-β production by mouse macrophages, and IL-10 has suppressed bone marrow-derived TGF-β (42, 43).

Significantly, adding IL-2 and or TNF-α to SLE lymphocytes or antagonizing IL-10 did not normalize active TGF-β production in SLE. Thus, decreased TGF-β production did not appear to be secondary to other obvious cytokine defects. Other mechanisms need to be identified.

Recently, it has been reported that murine peritoneal macrophages use plasmin bound to the cell surface to convert latent to active TGF-β (44). Human monocytes and NK cells appear peculiarly suited to converting TGF-β to its active form by plasmin activity, since these cells constitutively express urokinase plasminogen activator receptors on their cell surface. T cells, by contrast, only express these receptors after activation (45). Of interest, plasminogen activator activity is decreased in SLE (46, 47, 48). Thus, decreased conversion of latent to active TGF-β might be due to this decreased enzymatic activity.

TGF-β can have beneficial or deleterious effects in lupus. SLE-like autoantibodies appear in TGF-β knockout mice (49), and the introduction of TGF-β genes into the skeletal muscle of the lupus-prone MRL/lpr mice decreased autoantibody production (50). Recently TGF-β has been reported to promote the growth of murine CD4+ cells and CD8+ cells (51, 52). With human peripheral blood cells, we have found that TGF-β costimulates CD8+ T cells to develop down-regulatory activity (4, 10). TGF-β is also an immunosuppressive cytokine, as it inhibits T and B cell proliferation, NK cell cytotoxic activity, and the generation of T cell cytotoxicity (5, 53, 54, 55). As stated above, TGF-β complexed to IgG can be immunosuppressive (39, 56).

The net biologic effect of TGF-β is determined by the local cytokine concentration and the cell types affected. Whereas picogram quantities of TGF-β are needed for suppressor cell induction (10), nanogram amounts are generally required for suppressive effects 53–55). De Jong et al. showed that TGF-β had costimulatory activity for naive CD4+CD45RA+ cells but suppressed the proliferation of activated or memory CD4+CD45RO+ cells (57). Thus, TGF-β may promote the maturation of immature cells and have the opposite effect on mature cells. TGF-β may promote the maturation of immature cells and have the opposite effect on mature cells. TGF-β production in the kidneys contributes to the chronicity of glomerulonephritis (58).

It is well established that SLE patients lack regulatory cells capable of controlling B cell hyperactivity (2). Because both TGF-β and IL-2 production are decreased in SLE, and this combination of cytokines can induce down-regulatory T cell function in healthy individuals (10), we have asked whether these cytokines can reconstitute regulatory cell function. This remarkable inhibition of spontaneous Ig synthesis in two individuals with active SLE after exposure of their PBMC to TGF-β and IL-2 is an encouraging preliminary result.

We thank Drs. Alain Bernard, William Stohl, Bruce Pratt, Jay Unkeles, and Satwant Narula for providing us with useful mAbs. We also thank Ms. Lillie Hsu for her expert technical assistance and Dr. Francisco Quismorio, Jr. for help in patient acquisition. We are grateful to Dr. Daniel Rifkin for providing the genetically engineered mink lung cells for the TGF-β assay.

1

This work was supported by grants from the National Institutes of Health (AR 29846 and AI 41768), from the Nora Eccles Treadwell Foundation, and from Schering Plough Pharmaceuticals.

3

Abbreviations used in this paper: SLE, systemic lupus erythematosus; MLEC, mink lung epithelial cells.

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