Bi-directional modulation of T cell-dependent antibody production by prostaglandin E2
Xiaowen He1,2,
Cornelia M. Weyand3,
Jörg J. Goronzy3,
Wanyun Zhong3 and
John M. Stuart1,2
1 Research Service 151, VA Medical Center Memphis, 1030 Jefferson Avenue, TN 38104, USA
2 Department of Medicine, University of Tennessee, Memphis, TN 38104, USA
3 Department of Medicine, Mayo Clinic and foundation, Rochester, MN 55905, USA
Correspondence to:
X. He
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Abstract
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T cell-dependent Ig production involves interaction between T cells and B cells. This study evaluated the effects of prostaglandin (PG) E2 on Ig production in a system in which B cells were co-cultured with autologous CD4+ T cell clones non-specifically activated by anti-CD3. The effects of PGE2 on T cell-dependent Ig production differed substantially, depending on the T cells employed. We selected six T cell clones that were able to enhance Ig production (resistant T cell clones) and six T cell clones that inhibited Ig production in the presence of PGE2 (sensitive T cell clones) for comparison. The resistant T cells produced high levels (>1000 pg/ml) of IL-2 and/or IL-4, and expressed high CD40L, OX40 and CD45RA, and low CD45RO. In contrast, sensitive T cells secreted low IL-2 (<500 pg/ml) and IL-4 (<200 pg/ml), and expressed low CD40, OX40 and CD45RA, and high CD45RO. Adding supernatant derived from resistant T cell clones restored Ig production inhibited by PGE2, while removing IL-2, IL-4 or IL-10 using specific antibodies inhibited Ig production. In addition, we demonstrated a direct effect of PGE2 on B cells to enhance Ig production. Consistently, in the presence of resistant T cells, PGE2 increased B cell proliferation and differentiation. In conclusion, the effects of PGE2 on Ig production consist of its indirect effects through T cells and its direct effects on B cells. The outcome of the effects can be up-regulatory or down-regulatory, depending whether resistant or sensitive T cells are involved.
Keywords: B lymphocytes, cytokines, Ig, inflammatory mediators, T lymphocytes
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Introduction
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Development of an inflammatory response is a complex process involving vasoactive factors such as histamine, bradykinin, nitric oxide and prostaglandins (PG) (1). PG have been detected in many experimental and clinical inflammatory environments (2,3). However, direct injection of PG into the skin causes only minimal swelling, demonstrating that PG alone have little direct inflammatory capacity. In contrast, in the presence of other mediators, PG can amplify the local inflammatory response (4,5). Furthermore, inhibition of the synthesis of PG by pharmacological intervention is effective in down-regulation of acute inflammation and suppression of many of its symptoms.
In contrast to the effects of PG in modulating acute inflammation, their role in chronic inflammation is less clear. In adjuvant arthritis, an animal model of joint inflammation, high doses of PGE2 were found to suppress the inflammatory response (6). High levels of PGE2 have been found in synovial fluid and in cultures of synovial lining cells taken from rheumatoid arthritis patients (7); however, the destructive nature of the disease continues unabated even after prolonged treatment with PG-inhibiting drugs (8). Thus, the proinflammatory role of PG and the questions of whether inhibition of PG is harmful or beneficial in chronic inflammation remain in doubt.
In inflammatory diseases, especially chronic inflammatory diseases such as rheumatoid arthritis, acquired immunity plays a crucial role. T cell-dependent antibody production is believed to be important for pathogenesis. Therefore, studying the effects of PGE2 on T cell-dependent antibody production is essential for fully understanding the role of PGE2 in inflammation and the effects of down-regulation of PGE2 by pharmacological intervention.
T cell-dependent Ig production involves multiple steps of T and B cell collaboration (9,10). Two pivotal processes in the collaboration are the interaction of surface molecules on both cell types and the secretion of lymphokines by T cells. Among the interactive surface molecules, CD40 ligand (CD40L)CD40 and CD134 (OX40)CD134 ligand are believed to be important for delivering a Th cell contact signal to B cells (1115). The interaction of 4-1BB (CD137) and its ligand is also thought to regulate humoral immune responses, but its role is still under investigation (16,17).
In addition, multiple lymphokines promote Th cell and B cell activities, and are involved in T cell-dependent Ig production. Among them, IL-2, once considered to be strictly a growth factor for activated T cells, has been found to promote Ig production in the presence of CD40L (1821). IL-4 not only promotes B cell activation, proliferation and differentiation, but also promotes T cell proliferation and inhibits T cell apoptosis induced by cytokine withdrawal. The actions of IL-4 on T cells are believed to be mediated through a shared receptor signaling component that forms the
chain of the IL-2 receptor (19,2225). IL-10 is also an important T and B cell growth factor, especially in the presence of PGE2 (26,27).
In the present study, we studied the effects of PGE2 on T cell-dependent antibody production in a system in which B cells were co-cultured with autologous CD4+ T cell clones activated by anti-CD3 mAb. We found that the effects of PGE2 consisted of indirect effects through T cells and direct effects on B cells. The outcome of the effects depended on the particular T cells involved. PGE2 inhibited the Ig production induced by sensitive T cells, but enhanced the Ig production induced by resistant T cells. The sensitivity of the T cells to PGE2 was not associated with Th1/Th2 lymphokine secretion pattern, but correlated with the amount of IL-2 and IL-4 secreted, and with CD40L, OX40 and CD45 expression.
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Methods
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Generation and maintenance of human T cell clones
The details for generation and maintenance of human T cell clones have been described previously (28). In brief, mononuclear cells were purified from heparinized blood of a normal individual by Ficoll-Hypaque density-gradient centrifugation. Cells were cultured at 0.51x106 cells/ml for 24 h in culture plates coated with antibody to CD3. The stimulated cells were then distributed into 96-well microtiter plates with at an average concentration of 0.5 cells/culture in the presence of 1x104 irradiated filler cells and 50 U/ml recombinant IL-2 (PreproTech, Rocky Hill, NJ). After 1418 days, the outgrowing T cell clones were identified and cultured with irradiated filler cells at 3x105 cells/culture and 20 U/ml recombinant IL-2 in 24 well plates. After an additional 710 days, the emerging T cell clones were stained with FITC-conjugated antibody to CD4 and phycoerythrin (PE)-conjugated antibody to CD8 (Becton Dickinson, San Jose, CA), and subjected to flow cytometric analysis. CD4+CD8- T cell clones were selected and maintained by repeated re-stimulation every 710 days in the presence of 10 U/ml exogenous IL-2 and irradiated filler cells.
Purification of B cells
Mononuclear cells were isolated from heparinized blood samples by density-gradient centrifugation on Ficoll-Hypaque. The mononuclear cells at 5x106 cells/ml were incubated in serum-free RPMI 1640 with 50 mM L-leucine methyl esterHCl (Sigma, St Louis, MO) for 45 min at room temperature to eliminate the monocytes (29). After washing, T cells were depleted by rosetting with 2-AET (Sigma)-treated sheep red blood cells for 90 min and centrifugation on a Ficoll gradient. Non-rosetted cells were highly enriched in B cells. According to analyses by flow cytometry with antibodies to human CD3, CD14 and CD19 (PharMingen, San Diego, CA), the resulting cell population contained <5% CD3+ and <1% CD14+ cells.
T cellB cell co-cultures
Purified B cells at 5x103 cells/culture were co-cultured with cloned T cells at 1x105 in duplicates or triplicates in 96-well flat-bottom microtiter plates in the presence of anti-CD3. Culture supernatants were harvest after 14 days, and analyzed for IgM and IgG production by ELISA.
Determination of IgM and IgG
The concentration of total IgM and IgG in the culture supernatants was determined by ELISA. Maxisorb plates were coated with a polyvalent goat anti-human Ig (Sigma). Diluted culture supernatants were added and the plates incubated at 4°C overnight. After washing, second antibody consisting of peroxidase-conjugated goat anti-human IgM or anti-human IgG (Sigma) was added and incubation continued for 12 h. The plates were washed again, and developed with o-phenylenediamine dihydrochloride in substrate buffer consisting of citric acid, Na2HPO4, H2O and H2O2. Optical densities were measured at a wavelength of 490 nm with a reference wavelength of 650 nm. Standard solution of IgM and IgG were included in the assays.
Lymphokine analyses
T cell clones were stimulated with anti-CD3 at a concentration of 1x105 cells/culture in RPMI with 10% FCS in 96-well microtiter plates with flat-bottom wells. Cultures without any stimulation were set up in parallel to assess the background lymphokine secretion. Supernatants were harvested after 24 h. The cells and debris were removed from the supernatant by centrifugation. IFN-
, IL-2, IL-4, IL-5 and IL-10 were measured in duplicates using commercially available ELISA kits (Endogen, Cambridge, MA and R & D, Minneapolis, MN).
Flow cytometry analysis
To determine the expression of CD40L, OX40 and 4-1BB on T cells, the 1x105 cloned T cells/well were stimulated by immobilized antibody to CD3 with or without PGE2 at 10-6 M in 96-well microtiter plates with flat-bottom wells. For CD40L staining, cells were harvested after 8 h. For OX40 and 4-1BB, the cells were harvest after 44 h. The cells were pre-incubated with human IgG to block Fc binding and then stained with FITC-conjugated anti-CD40L, FITC-conjugated anti-OX40 or PE-conjugated anti-4-1BB in separate tubes (PharMingen).
To determine the expression of CD45RA and CD45RO expression, the T cells at resting stage were stained with PE-conjugated antibody to CD45RA or FITC-conjugated antibody to CD45RO (PharMingen).
To determine the expression of CD19 and CD138 (Syndecan-1) on B cells, 5x103 purified B cells/well were co-cultured with the 1x105 selected cloned T cells/well in the presence immobilized antibody to CD3 with or without PGE2 at 10-6 M. After 8 days, cells from each culture were harvested and evenly distributed into tubes, which were stained with both anti-CD19PE and anti-CD138FITC, or with anti-CD4PE.
As control, FITC- and/or PE-labeled mouse IgG1,
was used (PharMingen). The samples were analyzed by flow cytometry. The surface protein expression was assessed by the mean fluorescent intensity.
Statistical analysis
Statistical evaluation of differences was performed by using the unpaired t-test. When the normality test failed, the MannWhitney rank-sum test was used. Data were judged statistically significant when P < 0.05.
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Results
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Effects of PGE2 on Ig production depended on sensitivity of Th cells
We analyzed the action of PGE2 on T cell-dependent Ig production in a experimental system, in which B cells were co-cultured with autologous CD4+ T cell clones in the presence of immobilized anti-CD3. In this system, T cells were stimulated non-specifically by anti-CD3, to provide help, leading to B cell proliferation and Ig production in the absence of antigen. In the cultures without anti-CD3, no Ig production was detected. Thirty-seven CD4+ T cell clones were used. PGE2 at different concentrations was added at initiation of the culture. The effects of PGE2 on Ig production differed substantially among cultures, depending on the T cell clone used. PGE2 variously down-regulated, up-regulated or had no effects on Ig production. Examples the regulation of Ig production by two different T cell clones are shown in Fig. 1
. The effect of PGE2 was dose dependent and appeared to be maximal at 10-6 M.

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Fig. 1. IgM and IgG production in T cell/B cell co-cultures was modulated differently by PGE2 depending on the T cell clone used. T cells stimulated by anti-CD3 were co-cultured with purified B cells in the presence of PGE2 as described in detail in Methods. The results are expressed as mean ± SD of duplicate tests. IgM and IgG production induced by clone H63 was inhibited, while that induced by clone H48 was enhanced by PGE2.
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From 37 tested clones, six that inhibited Ig production in the presence of PGE2 (sensitive T cell clones) and six that enhanced Ig production (resistant T cell clones) were selected for further study. On average, in the absence of PGE2, the sensitive T cell clones induced 16.8 ± 7.6 µg/ml of IgM and 4.7 ± 2.2 µg/ml of IgG, as compared with 17.1 ± 12.3 µg/ml of IgM and 5.2 ± 3.1 µg/ml of IgG for resistant clones. However, in the presence PGE2 at the concentration of 10-6 M, the IgM and IgG production induced by sensitive T cell clones was reduced to 1.6 ± 1.7 and 0.3 ± 0.2 µg/ml. In contrast, IgM and IgG production induced by resistant T cell clones increased to 45.9 ± 17.1 and 17.8 ± 6.2 µg/ml respectively (Fig. 2
).

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Fig. 2. Effects of PGE2 on T cell-dependent IgM and IgG production induced by either sensitive (A and C) or resistant (B and D) T cell clones. Cloned T cells stimulated by anti-CD3 were co-cultured with purified autologous B cells from a normal individual without PGE2 (black bars) or with PGE2 at 10-6 M (gray bars).
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Resistant T cells expressed high level of CD40L and OX40
The surface molecules CD40L, OX40 and 4-1BB are believed to be involved in the regulation of T cell-dependent Ig production. We, therefore, studied the effects of PGE2 on these markers in the two groups of clones. The six sensitive and six resistant clones were each stimulated with immobilized anti-CD3 with or without PGE2, harvested, stained with FITC-labeled antibodies and analyzed by flow cytometry at the time points when each of these surface proteins reached peak expression (15,16,30). On average, PGE2 inhibited CD40L and OX40 expression. The resistant T cell clones expressed significantly more of both molecules than sensitive ones either in the absence or in the presence of PGE2. There was no substantial difference in 4-1BB expression between the two groups of the T cells in the absence of PGE2. However, the expression of the 4-1BB in the resistant clones was significant higher than in the sensitive clones in the presence of PGE2 (Table 1
).
Lymphokine secretion and CD45 isotype expression were correlated with Ig production in the presence of PGE2
We have previously found that sensitivity of T cell proliferation to PGE2 is correlated with lymphokine secretion profile and CD45 isotype expression (28). Therefore, we tested the possibility that antibody secretion may also be related to these parameters by comparing the lymphokine production and CD45 isotype expression between the two groups of T cell clones, which differ in modulation of Ig production. Table 2
shows the lymphokines secreted by the two groups of CD4+ T cell clones after anti-CD3 stimulation in the absence and presence of PGE2. As Table 2
shows, in the absence of PGE2, the T cell clones from resistant cultures secreted high levels (>1000 pg/ml) of IL-2, IL-4 or both, whereas the T cells from sensitive cultures secreted low levels of both IL-2 (<500 pg/ml) and IL-4 (< 200 pg/ml). In addition, almost all T cell clones from resistant cultures secreted all of the lymphokines tested, including IL-10, while the sensitive clones usually secreted fewer lymphokines. In the presence of PGE2, most of the lymphokine secretions are completely suppressed in sensitive T cell clones, whereas the lymphokines secreted by resistant T cell clones are diminished but still present. No correlation of Th1 or Th2 lymphokine pattern with the sensitivity of their helper function to PGE2 was found.
CD45 isoform expression of the two groups of the T cells was analyzed by flow cytometry. The mean intensity of CD45RA expression on the sensitive T cell clones was significantly lower than that on resistant clones. In contrast, CD45RO expression was significantly higher on sensitive clones (Table 3
). These data indicated that the sensitive and resistant T cells as identified by T cell-dependent Ig production in the presence of PGE2 are also characterized by distinct lymphokine profiles and CD45 isotype expression.
Effects of culture supernatant and anti-lymphokine antibodies on the Ig production in the presence of PGE2
Further experiments were conducted to determine whether the supernatants from activated resistant T clones could restore PGE2 inhibited Ig production. In these experiments, supernatants were collected from resistant T cell cultures that were stimulated by anti-CD3 for 24 h and added to cultures with sensitive clones in the presence of PGE2 at concentration of 10-9 to 10-5 M. The supernatants increased IgM in these cultures (Fig. 3
). In comparison, the supernatant derived from the sensitive T cell clone, H63, had no significant effect on the IgM production induced by resistant T cell clone, either H20 or H48, in the presence of PGE2 (data not shown). These data support the view that T cell lymphokines are pivotal in the T cell-dependent Ig production in the presence of PGE2.

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Fig. 3. Up-regulation of PGE2 inhibited Ig production by supernatant derived from resistant T cells. B cells at 1x104 cells/culture were co-cultured with a sensitive T cell clone, H63, at 1x105 cells/culture. The cultures were set up either with culture medium (dark bars) or with supernatants derived from a PGE2-resistant T cell clone, H20, previously stimulated by anti-CD3 for 24 h (gray bars). PGE2 was added at initiation of the culture. IgM concentrations were measured after 14 days of culture by ELISA and are expressed as mean ± SD of triplicated tests. Two PGE2-sensitive lines (H63 and H64) were each tested in two separate experiments and showed similar results, but only the data from clone H63 is shown. In this and following figures IgG was also analyzed and showed parallel results, but only IgM secretion is shown for simplicity.
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The role of individual lymphokines in T cell-dependent Ig production was further analyzed by using neutralizing antibodies. In these experiments, anti-IL-2, anti-IL-4 or anti-IL-10 was added into the cultures consisting of B cells and resistant T cell clones stimulated by anti-CD3. None of the antibodies had a clear impact on the IgM production in the absence of PGE2. However, each of the neutralizing antibodies reduced Ig production when PGE2 was present (Fig. 4
). These data suggested that that all of the three lymphokines were important for T cell-dependent Ig production in the presence of PGE2.

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Fig. 4. Down-regulation of Ig production by anti-IL-2, anti-IL-4 or anti-IL-10 in the presence of PGE2. B cells at 1x104 cells/culture were co-cultured with an anti-CD3 stimulated PGE2 resistant T cell clone, H20, at 1x105 cells/culture without PGE2 () or with PGE2 at 10-6 M ( ). Anti-IL-2, anti-IL-4 or anti-IL10 was added at initiation of the cultures and repeated after 7 days. IgM (mean ± SD) was measured after 14 days of culture by ELISA. Data presented are representative of two independent experiments with similar results.
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PGE2 acts directly on B cells to enhance Ig production
PGE2 inhibits the proliferation of almost all T cells and inhibits that of resistant T cells to a lesser extent (28). In comparison, PGE2 enhanced Ig production induced by resistant CD4+ T cell clones, suggesting that the existence of a direct effect of PGE2 on B cells, which promotes B cell secretion of Ig in the presence of resistant T cells. To determine the direct effects on B cells, resistant T cell clones H35 and H20 were used. B cells, T cells or both were pre-incubated either with PGE2 or with medium for 5 h and extensively washed before the T cell/B cell co-cultures were set up. Experiments with both T cell clones showed similar results in the separate experiments. The results with T cell clone H35 are shown in Fig. 5
. Pre-incubation of B cells with PGE2 enhanced Ig production. In contrast, pre-incubation of T cells with PGE2 led to suppression. Pre-incubation of both B and T cells resulted in Ig production that was intermediate between the two extremes. These data demonstrated that there is a direct enhancing action of PGE2 on B cells and the outcome of the action of PGE2 on Ig production depends on a balance between its enhancing action on B cells and its ability to down-regulate cytokine production by T cells.

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Fig. 5. Enhancement of Ig production by a direct effect of PGE2 on B cells. B cells or cloned T cells (H35) or both were pre-incubated with PGE2 at a concentration of 10-6 M or with complete culture medium for 5 h. After extensive washing, B cells at 5x103 cells/culture were cultured with cloned T cells at a concentration of 1x105 cells/culture as indicated in the presence of anti-CD3. IgM (mean ± SD) was measured after 14 days of culture by ELISA. Data presented are representative of two independent experiments with similar results using T cell clone H35 and H20.
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PGE2 enhanced B cell proliferation and differentiation
A further experiment was carried out to study the effects of PGE2 on B cell proliferation and differentiation in the presence of resistant T cells using T cell clone H35 and H20. In these experiments, purified B cells at 5x103 cells/culture were co-cultured with cloned T cells at 1x105 cells/culture stimulated by anti-CD3 in the absence or presence of PGE2 at 10-6 M. After 8 days, the cells in the culture were counted under a microscope using Trypan blue exclusion to determine viability. In the culture with clone H35, in the absence of PGE2, the total number of cells increased to 1.8x105/culture, while in the presence PGE2 the cells increased to 1.2x105/culture. The expression of CD19, CD138 and CD4 were determined by flow cytometry. In the control cultures without PGE2, CD138+ cells accounted for 0.7%, CD19+ cells accounted for 13% and CD4+ cells were 85% of the total cells (Fig. 6A and C
). In contrast, in the presence of PGE2 at 10-6 M, the CD138+ population increased to 8%. The CD19+ cells were increased, but their number could not be precisely determined, because they merged with the T cell population (Fig. 6B
). This phenomenon might be caused by decrease or loss of the CD19 marker at this stage of B cell differentiation. However, it was estimated that the CD4+ T cells were 49%, indicating that B cells, including CD138+ cells, accounted for ~50% of total cells (Fig. 6D
). Therefore, the total B cell number increased from the original 5x103 to 2.5x104 in the control culture without PGE2 and to 6.0x104 in the culture with PGE2. The TB cell co-cultures with T cell clone H20 had similar results (data not shown). The TB co-cultures with the sensitive T cell clones H63 and H64 were tested in parallel. The total number of the cells in the cultures with PGE2, including both T cells and B cells, was reduced to <1x104 cells/culture after 8 days. These data demonstrated that PGE2 enhanced B cell proliferation and differentiation even in the presence of resistant clones.

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Fig. 6. B cell proliferation and differentiation in T cell co-cultures. B cells at 5x103 cells/culture were cultured with cloned T cells (H35) at a concentration of 1x105 cells/culture in the presence of anti-CD3 and in the absence (A and C) or present (B and D) of PGE2 at 10-6 M. After 8 days, cells were harvested and stained with PE-labeled anti-CD19 and FITC-labeled anti-CD138 (A and B) or with PE-labeled anti-CD4 (C and D). The cells were analyzed by flow cytometry. The experiments were also conducted using T cell clone H20, which produced similar results but for simplicity only the data for H35 are shown.
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Discussion
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Our results revealed that the effects of PGE2 on Ig production depend on the T cells involved. PGE2 inhibits Ig production induced by sensitive T cells and up-regulates Ig production induced by resistant T cells. The resistant T cells secrete high levels of IL-2 and/or IL-4, and exhibit high CD45RA and low CD45RO expression, whereas sensitive T cells secrete low levels of both IL-2 and IL-4, and have low CD45RA and high CD45RO expression.
In the 1990s, it was reported that, in mice, PGE2 and increased intracellular cAMP inhibit lymphokines production and proliferation of Th1 cell, but not Th2 cells (3133). In humans, typical Th1 and Th2 cells are raremost T cells secrete both Th1 and Th2 lymphokines and can be characterized as Th0 type (34). Working with human lymphocytes, some studies have shown that increased intracellular cAMP or PGE2 modulates T cell lymphokine production in a way similar to its action on murine cells (35,36). However, the effects of PGE2 on T cell activities, such as T cell proliferation and T cell help, have not been established. In a recent study, we found that the effect of PGE2 on T cell proliferation did not relate to the Th1/Th2 lymphokine patterns of T cells, but to the amount of IL-2 and/or IL-4 secreted. PGE2 selectively inhibited the proliferation of the T cells that secreted low levels of both IL-2 and IL-4 (28). Consistent with this observation, the present study demonstrated that resistant and sensitive T cells, as measured by Ig production, share similar features.
The resistance of T cells to PGE2 relates to the amount of lymphokine production. In the presence of PGE2, most lymphokine secretion is completely suppressed in sensitive T cell clones, whereas the lymphokines secreted by resistant T cell clones are diminished but still present. The residual lymphokines in the resistant T cells are sufficient to maintain cell function. We further observed that supernatant from resistant T cell clones enhances Ig production induced by sensitive T cell clones, whereas elimination of IL-2, IL-4 or IL-10 from culture supernatant by specific antibodies inhibits Ig production induced by resistant T cells in the presence of PGE2.
We have recently shown that either IL-2 or IL-4 is sufficient to restore T cell proliferation inhibited by PGE2 (28). In contrast, neither IL-2 nor IL-4 nor IL-10 are able to restore PGE2 inhibited Ig production and adding a combination of IL-2, IL-4 and IL-10 were less effective in restoring Ig secretion in the presence of PGE2 than culture supernatant from resistant clones (data not shown). These data indicated that the Ig production in the presence of PGE2 requires the cooperative interaction of multiple lymphokines and favor the possibility that other lymphokines are also involved. In this regard, several lymphokines have been shown to promote T cell and/or B cell activities. For example, IL-9 and IL-15 promote T cell activities (19,24), IL-5 and IL-13 promote B cell activities (37,38), while IL-6 and IL-7 promote the activities of both T cell and B cells (19,37,39,40). Some of these lymphokines are produced by T cells but most are not. In our experimental system, individual T cell clones secreted a broad spectrum of lymphokines. In vivo, however, this might also be achieved by cooperation among cells, including non-T cells. Identification of every lymphokine involved in T cell-dependent Ig production is beyond the scope of the present study.
It is of interest that the expression of CD45 isotypes, which are believed to be markers for the memory status of the cells, can be linked to the lymphokine production and the sensitivity of T cells to PGE2. While the exact function of the CD45 isoforms is not clearly understood, it is known that naive cells express the CD45RA isoform. After activation some cells express CD45RO, leading to the suggestion that CD45RO is a marker for memory T cells (41). However, some in vivo studies have modified this view and demonstrated that the activated cells can revert to expression of CD45RA (4244). Therefore, the resistant T cell clones might derive from the CD45RA+ naive and reverted memory T cells. To confirm the correlation between CD45 isotype expression and amount of lymphokine production, we tested the IL-2 and IL-4 production of isolated CD45RA+ T cells and CD45RO+ T cells from peripheral blood of normal individuals in the presence of anti-CD3. Consistent with previous studies, we found that freshly isolated CD45RA+ T cells usually produce only very small amounts of IL-2 and IL-4 (41). However, these cells become potent lymphokine producers and remain CD45RA+ after a few cycles of in vitro stimulation. In comparison, CD45RO+ cells secreted less IL-2 and IL-4 (data not shown).
There is substantial evidence indicating that CD40L and OX40 expression is crucial for T cell-dependent Ig production (1115). 4-1 BB is also believed to be involved in the TB cell interaction (16,17). We examined these surface molecules at the time points when their expression reached its peak. We found that the resistant clones expressed significantly higher CD40L and OX40 than sensitive clones in the absence and presence PGE2, and expressed higher 4-1BB only in the presence of PGE2. In contrast, after 20 h of culture, there was no clear difference between resistant clones and sensitive clones in CD40L expression in the absence and presence of PGE2 (data not shown). Therefore, the higher expression of CD40L on resistant clones might happen only at its peak expression. Considering resistant clones are better helpers for B cells, these data seem to suggest that these molecules play positive roles in the TB cell interaction in the presence of PGE2. Recently, other studies have demonstrated that ligation of CD40 by anti-CD40 antibody or CD40L influences Ig production both positively and negatively. The effects depend on the density of CD40L as well as the stage of B cell activation and differentiation (21,45,46). If it is the case, the function of the surface molecules seems to be complex and more study will be needed to understand the significance of the current observation.
Our data showed a direct effect of PGE2 on B cells, which promotes B cell proliferation, differentiation and Ig production. These data are consistent with the study from Garrone et al., who have studied the effects of PGE2 on B cells by utilizing an experimental system in which T cell action was replaced by anti-CD40 mAb and lymphokines. They demonstrated that the direct action of PGE2 on B cells results in enhancement of IL-4- and IL-10-dependent B cell proliferation and IL-10-induced Ig secretion (27). Interestingly, the direct positive effects only function in the presence of resistant T cells, which can survive and provide B cell help even in the presence of PGE2. In the culture with sensitive T cells, PGE2 inhibited both T cells and B cells. Therefore, the effects of PGE2 on Ig production depend on the balance of its indirect negative effect through T cells and a direct positive on B cells. Among them, the Th cell is by far the most important and the basis for the positive effect of PGE2 on B cells.
These studies demonstrate how PGE2 regulates T cell-dependent Ig production. Through extrapolation to in vivo situation, it is speculative that PGE2 exerts different effects on Ig production in different phases of the inflammatory process. In the acute phase of inflammation, activated naive or reverted memory T cells are involved. High production of lymphokines by activated T cells leads to up-regulation of antibody production that is enhanced by the presence of PGE2. The inhibition of PGE2 at this stage will lead to the reduction of the inflammatory response and to down-regulation of antibody production. However, in chronic inflammation, T cells have experienced multiple cycles of doubling, become low lymphokine producers and are CD45RO+. At this stage, they become sensitive to PGE2. The presence of PGE2 will lead to a reduced level of antibodies. Thus, the ability of PG inhibition to reduce chronic inflammation will be limited.
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Acknowledgments
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This work is supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, Memphis, TN.
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Abbreviations
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CD40L CD40 ligand |
PE phycoerythrin |
PG prostaglandin |
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Notes
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Transmitting editor: S. L. Swain
Received 31 May 2001,
accepted 9 October 2001.
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