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The Journal of Immunology, 1998, 160: 1789-1795.
Copyright © 1998 by The American Association of Immunologists

Pentoxifylline Inhibits Ig {kappa} Gene Transcription and Rearrangements in Pre-B Cells1

Weihong Wang*, Satyajit Rath{dagger}, Jeannine M. Durdik{ddagger} and Ranjan Sen2,*

* Rosenstiel Research Center and Department of Biology, Brandeis University, Waltham, MA 02254; {dagger} National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India; and {ddagger} Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pentoxifylline (PF) has been used in a wide variety of clinical situations; however, the molecular consequences of this drug are not well characterized. In this paper we assayed the effects of PF in two models of pre-B differentiation. In 70Z pre-B cells, transcriptional induction of rearranged Ig {kappa}-chain gene in response to LPS was suppressed by PF, without affecting the induction of Rel family proteins. In contrast, {kappa} induction by IFN-{gamma} was not suppressed by PF, indicating that the drug inhibited certain activation pathways. We also found that LPS-induced activation of germline {kappa} transcription and V{kappa} to J{kappa} recombination were inhibited by PF in the pre-B cell line 38B9. These observations suggest that PF may adversely affect B lymphopoiesis during chronic administration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pentoxifylline (PF)3 is clinically used as an anti-inflammatory drug and for the treatment of endotoxic shock. Its efficacy in these situations has been attributed primarily to the inhibition of TNF-{alpha} production by monocytes and macrophages (1). However, there is considerable evidence that PF affects many different cell types, including lymphocytes (2, 3, 4, 5, 6). Despite its clinical relevance, surprisingly little is known about the molecular mechanisms of PF action. We initiated a study of this drug (7) because of a proposed connection with the regulation of the transcription factor, nuclear factor-{kappa}B (NF-{kappa}B). We found that PF inhibited the induction of c-Rel protein, but not other NF-{kappa}B family members such as RelB and p65, in activated T lymphocytes. The effect was remarkably specific in that several other inducible transcription factors known to be important for T cell activation, such as nuclear factor of activated T cells (NF-AT) and AP-1, were unaffected. Transcription factor dysregulation was reflected in the suppression of IL-2 gene induction, but there was no effect on the activation of the IL-2R {alpha}-chain (IL-2R{alpha}) gene. Because both promoters have been implicated as targets of NF-{kappa}B proteins, these observations were consistent with the promoter-specific use of Rel family proteins. We proposed that c-Rel was required for IL-2 gene induction, whereas p50/p65 was likely to be the functional IL-2R{alpha} promoter factor. In contrast to the effects seen in T lymphocytes, PF treatment during B cell activation did not affect NF-{kappa}B induction, suggesting that B and T lymphocytes responded differently to this drug.

NF-{kappa}B induction has been implicated in the differentiation of pre-B cells to B cells. Most pre-B cell lines have very little nuclear NF-{kappa}B whereas mature B cells contain constitutively nuclear NF-{kappa}B. Induction of NF-{kappa}B during the pre-B to B cell transition has been proposed to play a role in the activation of the Ig {kappa} light chain locus, via its positive effect on the {kappa} intron enhancer (8, 9). For example, LPS induction of {kappa} gene expression in 70Z cells, or {kappa} gene recombination in 38B9 cells, has been shown to be NF-{kappa}B dependent (10). However, it must be noted that recent gene disruption experiments have raised doubts about the importance of the {kappa} enhancer/NF-{kappa}B system during B cell differentiation. Specifically, disruption of several NF-{kappa}B family genes (11, 12, 13, 14), such as p50, p65, and c-Rel, did not show significant effects on B cell differentiation. These result may be explained by postulating functional redundancy among Rel proteins; that is loss of p65, for example, may be functionally compensated by another Rel protein such as c-Rel. Conversely, genetic deletion of the {kappa} intron enhancer (and thereby the only known NF-{kappa}B-dependent regulatory sequence in the {kappa} locus) also had only a small decrease in the generation of {kappa}-producing B cells (15). Interestingly, those cells that did produce {kappa} light chains expressed this protein at equivalent levels as wild-type (enhancer-containing) cells. Therefore, the {kappa} enhancer is not essential for B cell ontogeny. However, the decreased numbers of {kappa}-producing cells suggests that the {kappa} intron enhancer contributes to the efficiency of {kappa} gene rearrangement.

In this paper, we investigated whether PF affects NF-{kappa}B-dependent transcription and recombination in pre-B cells. We found that LPS-induced {kappa} expression in 70Z cells was blocked by PF without any discernible effect on the induction of Rel proteins. Inhibition occurred at the level of {kappa} mRNA production. In 38B9 cells, LPS-induced {kappa} germline transcription as well as {kappa} recombination was inhibited by PF, again without affecting NF-{kappa}B induction. However, induction of {kappa} transcription by IFN-{gamma} was not suppressed by PF. Because IFN-{gamma}-mediated {kappa} gene induction proceeds by an NF-{kappa}B-independent pathway (16), our results are consistent with postinduction modulation of NF-{kappa}B activity resulting in decreased {kappa} transcription and recombination. Furthermore, these observations suggest a possible deleterious effect of PF on B cell lymphopoiesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and treatment

70Z/3 and 38B9 pre-B cell lines were grown in RPMI 1640 medium supplemented with 10% inactivated FBS plus penicillin and streptomycin. ß-Mercaptoethanol was included in the growth medium at a concentration of 5 x 10-5 M. LPS (Difco Laboratories, Detroit, MI) activation was conducted at a concentration of 10 µg/ml for the times indicated. PF (Sigma Chemical Company, St. Louis, MO) was used at various concentrations as indicated in the figure legends.

D10.G4.1 (Th2) cells were stimulated with 2 µg/ml Con A for 24 h and the Con A was neutralized by addition of {alpha}-methylmannoside (9 mM). Supernatant from activated cells were used in several experiments and is referred to as Th2 filtrate. The concentration of IL-4 in the filtrate was estimated at 200 U/ml against a standard curve using rIL-4 and CTLL-2 cells. rIL-4 and IFN-{gamma} were obtained from Genzyme Corp. (Boston, MA). The anti-IL-4 mAb, 11B11, was purified over an anti-rat Ig column from hybridoma culture supernatants and used at a final concentration of 3 µg/ml in culture.

Cell staining and analysis

The reagents used for staining were: goat anti-mouse {kappa}-fluorescein (Southern Biotechnology, Birmingham, AL); mAb187.1 (rat anti-mouse Ig{kappa}; American Type Culture Collection, Bethesda, MD; HB58) labeled with either biotin or fluorescein; mouse anti-rat Ig{kappa} fluorescein (MAR18.5, TIB216); avidin-fluorescein (Vector, Burlingame, CA). Cells stained with goat anti-mouse Ig Fc-fluorescein (Cappell, West Chester, PA), mouse anti-rat Ig{kappa} fluorescein or avidin-fluorescein were utilized as negative controls. Cells were fixed in 0.5% paraformaldehyde before analysis on a Coulter Epics 752 (Coulter, Hialeah, FL) or a FACScan. A total of 10,000 cells were counted, and data were analyzed with either EASY2 or Consort 30 software.

Protein assays

Preparation of nuclear extracts, electrophoretic mobility shift assays (EMSA), and Western blotting was conducted as previously described (7). EMSA probe for NF-{kappa}B was the H2K sequence element (17). Anti c-Rel (SC71) and Rel B (SC226x) antisera were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at 1:500 dilution for immunoblotting.

RNA analysis

Total cellular RNA was isolated using Ultraspec procedure (Biotecx Laboratories, Houston, TX) for experiments shown in Figures 2Go and 6Go. Cytoplasmic RNA was prepared as described by Maniatis et al. (18) for the experiments shown in Figure 4Go. For Northern blot analysis, RNA was fractionated through 1% agarose gels containing formaldehyde, transferred to nitrocellulose filters, and hybridized with the following 32P-labeled probes: 0.4 kb PstI fragment containing C{kappa} derived from pES202, 0.5 kb SacI-KpnI fragment derived from exon II of ß2-microglobulin, and 0.8 kb fragment derived from the rat GAPDH gene.



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  		FIGURE 2. Analysis of {kappa} mRNA expression. A, 10 µg of cytoplasmic RNA obtained from 70Z cells treated as indicated above each lane were fractionated through formaldehyde-containing agarose gels, transferred to nitrocellulose filters, and hybridized with a C{kappa} probe. Position of the 1.2-kb {kappa} mRNA is indicated by the arrow. All treatments were for 18 h. The blot was reprobed for GAPDH expression for normalization (lower panel). B, Dose-dependent suppression of LPS-mediated Ig{kappa} mRNA induction in 70Z cells. Cells were treated as indicated for 60 h and total RNA was analyzed by Northern blotting with a C{kappa} probe (marked {kappa}) or a ß2-microglobulin probe (marked ß2).

 


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  		FIGURE 6. Comparison of PF and IL-4 on {kappa} expression induced by IFN-{gamma}. 70Z cells were stimulated with 100 U IFN-{gamma} alone (A) or in the presence of rIL-4 (B) or PF (C). After 60 h of stimulation, cells were stained for Ig{kappa} expression (dotted line). Negative controls are shown in the solid lines.

 


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  		FIGURE 4. Analysis of Rel protein in 70Z cells treated with PF. A, EMSA using nuclear extracts from 70Z cells treated as indicated above the lanes and a radioactive NF-{kappa}B probe derived from the MHC class I gene, H2K. Position of NF-{kappa}B nucleoprotein complex is marked. B and C, Nuclear extracts as indicated were fractionated by SDS-PAGE, proteins transferred to nitrocellulose membranes that were probed using c-Rel (B)- or Rel B (C)-specific antisera. Positions of the respective proteins are indicated.

 
Germline {kappa} transcripts were detected as previously described (19) with minor modifications. Three micrograms of total RNA (Ultraspec) from 38B9 cells was reverse transcribed (dNTPs 1.5 mM, 0.2 µM J{kappa}2 primer, 20 U AMV reverse transcriptase (Boehringer Mannheim, Indianapolis, IN), 0.25 µl RNAsin) in a final volume of 20 µl at 42°C for 1 h. The reaction was diluted to 200 µl with TRIS-EDTA and 4 µl taken for PCR amplification using J{kappa}2 and {kappa}o primers. PCR reactions contained 0.2 mM dNTPs, Vent polymerase (New England Biolabs, Beverly, MA) in a final volume of 100 µl. A total of 28 cycles of 1 min denaturation at 94°C, 1.5 min annealing at 52°C, and 1.5 min elongation were used with a final elongation time of 7 min at 72°C. Twenty microliters of the reaction was electrophoresed through 1% agarose gels, and blotted onto nylon membrane (ICN, Aurora, Ohio), which was probed with the C{kappa} probe described above.

Nuclear run-on assays were performed essentially as described by Greenberg and Ziff (20). Nuclei were prepared from 70Z cells treated with LPS in the presence, or absence, of PF for 18 h. After 30 min transcription in vitro, the nuclei were treated sequentially with DNase I (10 min) and proteinase K (30 min). RNA was purified by two rounds of phenol extraction and precipitated with isopropanol. Six micrograms of linearized pSP72 (negative control), a pSP72 derivative containing a 0.4-kb PstI fragment encoding C{kappa} sequences or a plasmid containing GAPDH sequences, were immobilized on Hybond nylon membrane and hybridized to radioactive RNA (5 x 106 cpm/ml). The membranes were washed extensively and analyzed by phosphor imager quantification.

V{kappa}-J{kappa} recombination assays

One microgram of sheared genomic DNA isolated from 38B9 cells was used in PCR reactions containing J{kappa}2 primer and degenerate V{kappa} primers as described (19). One-fifth of the reaction was electrophoresed through 1% agarose gels and blotted onto nylon membranes, which were probed with a 32P-labeled 2.8-kb HindIII fragment containing all five murine J{kappa} gene segments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PF suppresses {kappa}-chain expression in 70Z cells

70Z cells have been widely used as model system to study the differentiation of pre-B cells to B cells. These cells contain productively rearranged Ig heavy (µ) and {kappa} light chain genes, but express only the IgH gene. Stimulation of cells by various inducers such as bacterial LPS, phorbol ester, and IFN-{gamma} induces {kappa} gene transcription and subsequent expression of Ig at the cell surface. Activation of {kappa} transcription by the first two reagents has been proposed to proceed via an NF-{kappa}B-dependent mechanism, whereas that by IFN-{gamma} is apparently independent of NF-{kappa}B. We tested the effects of the drug PF, which has been implicated in dysregulation of NF-{kappa}B-dependent transcription, on {kappa} induction in 70Z cells.

Cells were treated with LPS in the absence, or presence, of PF and assayed for surface {kappa} expression by flow cytometry (Fig. 1GoA). LPS activation induced {kappa} expression as expected (Fig. 1GoA, panel B), which was significantly diminished in the presence of PF (Fig. 1GoA, panel D). Treatment of the cells with PF alone did not affect {kappa} expression (Fig. 1GoA, panel C). The effect of PF on {kappa} expression was most evident at PF concentrations greater than 100 µg/ml as shown in the dose-response curve in Figure 1GoB. Northern blot analyses showed that PF blocked {kappa} gene expression at the level of mRNA production (Fig. 2GoA). Equal loading of RNA was confirmed by reprobing the filter with a probe for the GAPDH gene. We further examined the effect of different amounts of PF on {kappa} mRNA production (Fig. 2GoB). Dose-dependent suppression of {kappa} mRNA was observed, with half-maximal suppression requiring 30 µg/ml PF. To rule out deleterious side effects of PF treatment on these cells, we evaluated cell viability and growth rate of 70Z cells in the presence of the drug. Cell viability as assessed by trypan blue exclusion was the same as untreated control cells (>95% viable cells over a 6-day treatment period) (data not shown). At the end of 6 days, total cell numbers were reduced twofold in the PF-treated samples compared with the untreated cells (data not shown). Because these cells grow rapidly (approximately 10 to 12 h doubling time), these observations suggest that PF has a small effect on 70Z cell growth. Most experiments reported in this paper were conducted after 18 to 24 h of PF treatment, when neither cell numbers or viability were affected.



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  		FIGURE 1. PF induces a decrease in surface Ig{kappa} expression on LPS-stimulated 70Z/3 cells. A, Cells were incubated with either medium alone (panel A), with 10 µg/ml LPS alone (panel B), with 300 µg/ml PF alone (panel C), or with LPS and PF (panel D) for 64 h and then stained for Ig{kappa}. The percentage of {kappa}-positive cells was in each panel: A = 0.7%, B = 45.6%, C = 1.2%, and D = 16.6%. The negative control curves were stained with FITC goat anti-mouse Fc. These are shown in each panel as dotted lines (A = 0.8% positive, B = 1.1% positive, C = 0.34% positive, and D = 4.7% positive). In each case, profiles of 10,000 cells are shown. B, PF suppresses Ig{kappa} induction on LPS-treated 70-Z/3 cells in a dose-dependent manner. Cells were incubated with either medium alone or with 10 µg/ml LPS in the presence of from 0 to 300 µg/ml of PF for 64 to 68 h. Anti-Ig{kappa} staining is shown in the solid line.

 
The decrease in {kappa} mRNA levels in cells activated in the presence of PF could be due to reduced gene transcription or increased mRNA degradation. To distinguish between these possibilities we assayed transcription rate of the {kappa} gene by nuclear run-on assays. 70Z cells were treated with LPS in the presence or absence of PF for 18 h before isolation of nuclei, which were used to synthesize radioactive RNA in vitro. C{kappa} RNA was quantitated by hybridization to excess C{kappa} DNA immobilized on nylon membranes. Compared with a GAPDH control, significant C{kappa} RNA was detected in nuclei prepared from LPS-treated 70Z cells (Fig. 3Go, first two bars). In contrast, the level of C{kappa} RNA was significantly diminished, compared with GAPDH, in nuclei of cells activated in the presence of PF (Fig. 3Go, third and fourth bars). Thus, {kappa} gene transcription is reduced approximately 10-fold in PF-treated 70Z cells.



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  		FIGURE 3. Nuclear run-on analysis of {kappa} gene transcription in PF-treated 70Z cells. 70Z cells were treated with LPS (10 µg/ml) alone or in the presence of 300 µg/ml PF for 18 h. Nuclei were isolated and "run-on" assays performed as described in Materials and Methods. Briefly, radioactive RNA synthesized in vitro was hybridized to excess plasmid DNA-containing GAPDH sequences (positive control), C{kappa}, or no insert (negative control). The counts retained were quantified by phosphor imager analysis and, after subtraction of the background, normalized to GAPDH expression in LPS only treated cells (which is assigned the value 100 (first bar)). The data shown represents one of two experiments conducted independently.

 
In addition, we evaluated a possible contribution of PF to {kappa} mRNA stability. 70Z cells were activated with LPS for 18 h followed by addition of actinomycin D alone or together with PF. Cellular RNA extracted at several subsequent times were assayed by Northern blots for C{kappa} transcripts. Up to 3 h following the termination of transcription, no differences in {kappa} mRNA levels were detected in the presence or absence of PF. Decreased viability of cells with extended actinomycin D exposure precluded analysis at longer time points. These observations suggest that stability of {kappa} mRNA is not significantly affected by PF. We conclude that the primary effect of PF is to reduce {kappa} gene transcription in 70Z cells.

To investigate whether the decrease of {kappa} mRNA was due to an effect on NF-{kappa}B, we examined NF-{kappa}B activation by EMSA. As previously demonstrated, NF-{kappa}B activity was strongly induced in 70Z cells treated with LPS (Fig. 4GoA, lanes 1 and 2). However, no decrease in NF-{kappa}B activity binding was detected in nuclear extracts derived from cells activated by LPS in the presence of PF (Fig. 4GoA, lane 3). Treatment of cells with PF alone was not sufficient to activate NF-{kappa}B binding (Fig. 4GoA, lane 4). It has been previously suggested that the NF-{kappa}B family member c-Rel may be the functional {kappa} enhancer (and thereby {kappa} gene expression) activating protein in these cells (21, 22). Because EMSA is skewed toward the detection of p50/p65 heterodimers, we examined the nuclear expression of c-Rel by immunoblotting. Nuclear c-Rel was induced after 4 h LPS treatment (Fig. 4GoB, lanes 1 and 2). However, c-Rel induction was not affected by PF (Fig. 4GoB, lanes 3 and 4). Thus, the difference in {kappa} mRNA production was not due to diminished c-Rel expression. In the NF-{kappa}B-deficient plasmacytoma S107, {kappa} expression and demethylation can be restored by stable expression of Rel B (23). These observations have suggested that Rel B may be necessary for {kappa} gene expression. We therefore evaluated Rel B expression in 70Z cells by immunoblotting. No differences were observed in the levels of nuclear Rel B in the presence, or absence, of PF (Fig. 4GoC). We conclude that inhibition of {kappa} mRNA occurs without any apparent change in the expression of Rel family proteins.

IL-4 and PF differently affect {kappa} gene induction by IFN-{gamma}

Several stimuli are known to induce {kappa} expression in 70Z cells. While most proceed via induction of NF-{kappa}B, IFN-{gamma} activation is of particular interest because it has been proposed to activate via an NF-{kappa}B-independent pathway (16). We assayed the effects of agents that suppress {kappa} expression on the alternative modes of {kappa} gene induction. IL-4 has been previously described to suppress LPS mediated {kappa} induction without significant effect on NF-{kappa}B DNA binding (24). LPS-mediated activation of cell surface {kappa} expression was reduced in a dose-dependent manner by a filtrate from an activated Th2 clone, D10.G4.1 (Fig. 5GoA). This effect was blocked by the anti-IL-4 mAb, 11B11, and reproduced by rIL-4 (data not shown). IL-4 also suppressed {kappa} induction at the mRNA level (Fig. 5GoB). Neither Th2 filtrate alone nor rIL-4 affected {kappa} mRNA production (Fig. 5GoB, lanes 1, 3, and 5). However, the strong induction of {kappa} mRNA by LPS (Fig. 5GoB, lane 2) was significantly reduced when activation was conducted in the presence of Th2 filtrate or rIL-4 (Fig. 5GoB, lanes 4 and 6, respectively). Thus, IL-4 and PF have similar effects on LPS-mediated {kappa} gene expression in 70Z cells.



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  		FIGURE 5. Effect of IL-4 on {kappa} mRNA expression in 70Z cells. A, Dose-dependent suppression of Ig{kappa} mRNA expression by supernatants from activated D10.G4.1 Th2 cells. 70Z cells were incubated in medium alone (A), 10 µg/ml LPS (B), or LPS with 0.03%, 0.1%, 0.3%, and 1% Th2 filtrate (C–F, respectively) for 60 h and then stained for Ig{kappa} expression. The negative control curves (dotted lines) were obtained by staining with FITC-avidin. B, RNA isolated from cells treated as indicated for 48 h was assayed by Northern blotting as described in the legend to Figure 2Go. D10 filtrate refers to supernatants from Con A-activated D10.G4.1 cells, and was used at a concentration of 1%. rIL-4 was used at 100 U/ml. 70Z cells were stimulated at a density of 3 to 5 x 105 cells/ml before being harvested for staining or RNA analysis.

 
However, a difference was noted between IL-4 and PF when we examined the effects of these reagents on the induction of {kappa} gene expression by IFN-{gamma}. Treatment of 70Z cells induced detectable levels of cell surface {kappa} expression (Fig. 6GoA). This induction was blocked significantly by rIL-4 (Fig. 6GoB), but not PF (Fig. 6GoC). These observations suggest PF selectively blocks only certain pathways of {kappa} gene induction.

PF suppresses sterile {kappa} transcription and gene rearrangements

Despite the value of 70Z cells as a probe for the pre-B to B cell transition, these cells represent a late pre-B cell stage because they have already undergone functional V{kappa} to J{kappa} recombination. To extend our observations with PF in 70Z cells, we assayed Abelson murine leukemia virus-transformed 38B9 cells. Because these cells undergo VH to DJH recombination in culture, and maintain their {kappa} loci in a predominantly germline configuration, they represent an earlier stage of pre-B cell differentiation compared with 70Z cells (25). However, LPS treatment of these cells induces NF-{kappa}B, germline {kappa} transcription, and {kappa} gene rearrangements (19).

In EMSA, NF-{kappa}B DNA binding was strongly induced in 38B9 cells treated with LPS (Fig. 7GoA, lanes 1 and 2), and PF treatment did not affect NF-{kappa}B induction, as observed previously in 70Z cells (Fig. 7GoA lanes 3 and 4). However, LPS induced sterile transcription of the germline {kappa} locus was markedly diminished in the presence of PF (Fig. 7GoB). Sterile transcription was assayed as previously described using specific primers to amplify cDNA synthesized from total cellular RNA, followed by electrophoretic separation and Southern blotting using a C{kappa}-specific probe. This procedure detects a transcript that is initiated 3.5 kb 5' of the J{kappa} gene segments and spliced to the C{kappa} exons (26). Untreated 38B9 cells do not contain detectable levels of this transcript, which was significantly induced after LPS treatment (Fig. 7GoB, lanes 1 and 2, marked by the arrow). LPS activation in the presence of PF decreased the germline transcript (Fig. 7GoB, lane 3) and PF treatment alone had no effect compared with untreated cells (Fig. 7GoB, lane 4). The results in Figure 7GoB were obtained using RNA from cells that had been activated for 18 h. Phosphor imager quantification of this data showed an approximately 90% decrease in the levels of germline {kappa} mRNA (data not shown). As a control, GAPDH mRNA was also amplified by PCR from the same samples (Fig. 7GoB, lower gel, labeled GAPDH). Analysis of RNA obtained from cells activated for 36 h showed a similar pattern of activation and suppression of germline transcripts (data not shown). We conclude that induction of germline {kappa} transcripts is also blocked by PF.



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  		FIGURE 7. Effect of PF on germline {kappa} transcription in 38B9 pre-B cells. A, Analysis of NF-{kappa}B activity in 38B9 cells. EMSA using nuclear extracts from 38B9 cells stimulated as indicated for 18 h, and the H2K {kappa}B element. Position of the major LPS-inducible complex is indicated. B, Germline {kappa} transcription was assayed by RT-PCR using cytoplasmic RNA prepared from 38B9 cells stimulated as indicated for 18 h. The products of RT-PCR were fractionated through 1% agarose gels, transferred to nylon membranes, and probed with 32P-labeled C{kappa} fragment as described in Materials and Methods. Position of the RT-PCR product is indicated by the arrow. Quantification of the band intensities by Phosphor Imager analysis showed an approximately ninefold decrease in the RT-PCR signal in the LPS + PF lane compared with the LPS alone lane. RT-PCR was also used to amplify GAPDH mRNA and the products were visualized by ethidium bromide staining after fractionation through 1% agarose gels (labeled as GAPDH).

 
In addition to assaying {kappa} locus transcription, the 38B9 cell line provides an opportunity to investigate the effects of PF on the induction of {kappa} gene rearrangements. These cells have been previously shown to undergo LPS-induced {kappa} recombination, which can be assayed by PCR (19). Genomic DNA prepared from 38B9 cells treated with LPS in the presence, or absence, of PF was used to assay {kappa} recombination. Linear conditions for the PCR assay were confirmed by using different amounts of genomic DNA to amplify the C{kappa} locus (Fig. 8GoA). Results of quantification by phosphor imager analyses are noted below the lanes. The strong induction of {kappa} rearrangement by LPS (Fig. 8GoB, lanes 1 and 2) was reduced when the activation was conducted in the presence of PF (Fig. 8GoB, lane 3). The data shown was obtained from cells that had been stimulated for 36 h with LPS; similar results were obtained with cells stimulated for 18-h. Phosphor imager quantification of the results indicated approximately 60 to 70% suppression of recombination. We conclude that PF inhibits germline transcription and gene rearrangements at the {kappa} locus.



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  		FIGURE 8. Effect of PF on {kappa} gene rearrangements. A, Linearity of the PCR assay was assessed by using serial dilutions of genomic DNA prepared from LPS-treated 38B9 cells as indicated above the lanes. After fractionation through agarose gels the PCR products were transferred to nylon membranes and probed with a radioactive J{kappa} probe. Phosphor imager quantification is noted below the lanes. B, Untreated 38B9 cells, or cells stimulated with LPS in the presence, or absence, of PF for 36 h were used to prepare genomic DNA. The {kappa} gene recombination was assayed by PCR using degenerate V{kappa} primers and a J{kappa} primer as detailed by Schlissel and Baltimore (19). PCR products were fractionated through 1% agarose gels, transferred to nylon membranes, and probed with a J{kappa} fragment. Position of V{kappa} J{kappa} recombined products is indicated. Phosphor imager quantification showed an approximately threefold decrease in the intensity of the recombined band in lane 3 compared with lane 2. Analysis of recombination after 18 h of activation gave similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper we show that activation of {kappa} gene transcription, which occurs during differentiation of pre-B cells to B cells, is inhibited by PF. Furthermore, reduced germline transcription of the unrearranged {kappa} locus is reflected in decreased V{kappa} to J{kappa} rearrangements. These results suggest that B lymphopoiesis may be affected during the clinical use of PF.

The {kappa} locus is regulated by two enhancers. One is located in the J{kappa}-C{kappa} intron (i{kappa}E) and the second 3' of the C{kappa} exons (3'{kappa}E) (27, 28). Several lines of evidence suggest that the activation of the rearranged {kappa} gene in 70Z cells, or the germline locus in 38B9 cells, is dependent upon the intron {kappa} enhancer. First, the intron enhancer is LPS inducible because of the presence of an NF-{kappa}B-binding site, whereas the 3'{kappa} enhancer is not LPS inducible. Because {kappa} transcription in both these cell lines is strictly LPS dependent, the {kappa} intron enhancer has been considered to be the critical regulatory target. Second, inhibition of NF-{kappa}B induction by the use of nondegradable I{kappa}B derivatives has been shown to prevent {kappa} transcription in both these cell lines (10). Because only the {kappa} intron enhancer, and not the 3'{kappa} enhancer, is regulated by NF-{kappa}B, it is likely that activation of the intron enhancer is the major regulatory event necessary for {kappa} transcription in these cells. Because induced {kappa} transcription is inhibited by PF in these cells, we propose that PF inhibits activation of the intron {kappa} enhancer. One other experimental result is consistent with this hypothesis. We also examined the effect of PF on the induction of germline {kappa} transcription in a pre-B cell line transformed by a temperature-sensitive abl oncogene (29, 30). When grown at the permissive temperature, these cells are phenotypically similar to standard Abelson retrovirus-transformed cell lines. Specifically, NF-{kappa}B and {kappa} gene expression is low. However, when shifted to nonpermissive temperature, NF-{kappa}B induction and germline {kappa} transcription occurs. However, the 3'{kappa} enhancer is not activated before, or after, temperature shift, suggesting that {kappa} transcription is dependent only on the intron enhancer (30). In these cells as well, we observed decreased germline {kappa} transcripts in the presence of PF (data not shown). Overall, we suggest that PF inhibits activation of the {kappa} intron enhancer.

A role for NF-{kappa}B in suppression of {kappa} gene expression by PF is also suggested by the inability of this drug to affect IFN-{gamma}-mediated {kappa} gene induction. In contrast to several other {kappa}-activating agents such as LPS and phorbol esters, which also induce NF-{kappa}B, IFN-{gamma} has been proposed to activate {kappa} transcription by an NF-{kappa}B-independent pathway (16). Our results indicate that PF suppresses {kappa} expression only when the gene is activated by an NF-{kappa}B-dependent mechanism. It is interesting to note that IL-4 appears to be a more general inhibitor of {kappa} transcription, blocking {kappa} gene induction by both NF-{kappa}B-dependent and NF-{kappa}B-independent pathways. In this context, it would appear that the partial suppression of LPS-induced NF-{kappa}B by IL-4 (24) may not be primarily responsible for inhibition of {kappa} expression.

The NF-{kappa}B-binding site in the {kappa} intron enhancer is a key determinant of enhancer activity. However, we found that induction of several Rel family proteins, including NF-{kappa}B (p50/p65), c-Rel, and Rel B, was unaffected by PF. One interpretation of these results is that the NF-{kappa}B site may not mediate PF-dependent suppression. Alternatively, it is possible that post-translational modification of Rel proteins in response to PF may alter their activating properties without affecting DNA binding. Although there is evidence for the phosphorylation of Rel proteins, the regulatory consequences of such modifications have not been well defined (31, 32). PF is a methyl xanthine derivative that is believed to function as a phosphodiesterase inhibitor, resulting in elevation of intracellular cAMP. However, effects of PF do not always correlate with increased cAMP. In particular, in 70Z cells cAMP has been shown to activate {kappa} light chain expression (33), whereas we found that PF inhibited {kappa} gene induction by LPS. Nevertheless, the possible intersection with the cAMP pathway is intriguing because of earlier studies that functionally link Rel proteins, such as dorsal and v-rel, with protein kinase A (34, 35). Although the mechanism of PF function in pre-B cells remains unclear at present, our findings reveal a potentially deleterious side effect of PF administration on B lymphopoiesis.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants GM 38925 to R.S. and GM 48691 to J.M.D. Back

2 Address correspondence and reprint requests to Ranjan Sen, Rosenstiel Research Center, MS029, Brandeis University, Waltham, MA 02254. Back

3 Abbreviations used in this paper: PF, pentoxifylline; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde phosphate dehydrogenase. Back

Received for publication April 30, 1997. Accepted for publication November 3, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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