Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Kuzin, I. I.
Right arrow Articles by Bottaro, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuzin, I. I.
Right arrow Articles by Bottaro, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

International Immunology, Vol. 13, No. 7, 921-931, July 2001
© 2001 Japanese Society for Immunology

Tetracyclines inhibit activated B cell function

Igor I. Kuzin1, Jennifer E. Snyder2,4, Gregory D. Ugine1, Dongming Wu1, Sang Lee3,4, Timothy Bushnell, Jr1, Richard A. Insel3, Faith M. Young1,2,3 and Andrea Bottaro1,2 1 Departments of Medicine,
2 Microbiology and Immunology, and
3 Pediatrics, and the
4 Graduate Program in Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA

Correspondence to: A. Bottaro, Immunology Unit, University of Rochester School of Medicine, URMC Box 695, 601 Elmwood Avenue, Rochester, NY 14642, USA


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Tetracyclines have recently been shown to exert a number of pleiotropic anti-inflammatory and immunomodulatory activities, independent of their antibiotic properties. These include the ability to inhibit metalloproteinases (MP), a class of enzymes involved in crucial cellular functions such as the shedding of soluble mediators and their receptors from the cell surface, as well as interaction with, and remodeling of, the extracellular matrix. Here we report that doxycycline at therapeutic concentrations (1–5 µg/ml) significantly suppresses Ig secretion and class switching by in vitro activated murine B cells. Suppression of Ig secretion correlates with a decrease in levels of mRNA for the terminal B cell differentiation-associated genes Blimp-1 and mad-4, as well as to a reduction in expression of the plasma cell markers Syndecan-1 and J chain. Inhibition of class switching occurs at the recombination stage and is also induced by other MP inhibitors, including tetracycline analogs lacking antibiotic activity and the chemically unrelated hydroxamate KB8301. These novel, direct effects of MP inhibitors on B lymphocytes suggest an intrinsic role for MP in B cell activation and likely explain some of the observed in vivo immunomodulatory properties of tetracyclines. Moreover, these findings have significant implications for tetracycline therapy in Ig-mediated autoimmune or allergic diseases and raise questions about the use of doxycycline-inducible transgenic systems for the study of B cell function.

Keywords: B lymphocyte, Ig, isotype switching, metalloproteinase, tetracycline


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Tetracyclines, among the most commonly prescribed antibiotics, also display a number of independent, pleiotropic activities on inflammatory and immune processes. They have been shown to specifically decrease levels of inducible NO synthase in activated macrophages by regulating the stability of its mRNA (1,2) and to inhibit phospholipase A2 (3). Cyclo-oxygenase-2-mediated prostaglandin synthesis is also differentially affected by various tetracyclines (4,5). Finally, tetracyclines can specifically inhibit the expression, activation from pro-enzyme precursor, as well as the enzymatic activity of metalloproteinases (MP) (6–8, reviewed in 9,10). MP inhibition is in part responsible for the tetracyclines' ability to modulate secretion of soluble factors released by proteolytic ectodomain shedding, such as tumor necrosis factor (TNF)-{alpha} (11) and Fas ligand (12), and to reduce the destruction of connective tissue during severe inflammation (reviewed in 9,10). This wide array of biological activities has resulted in the widespread application of tetracycline-based therapy in inflammatory disorders such as periodontitis and rheumatoid arthritis (7,13,14).

In a number of studies, tetracyclines have also been shown to decrease humoral immune responses in mice (15–17). Moreover, minocycline treatment reduced serum levels of rheumatoid factor and total Ig in rheumatoid arthritis patients (13,14). Tetracyclines have also been reported to suppress lymphocyte proliferation to mitogens and cytokine secretion in vitro, although the effect was variable and observed in only some systems (18–24). The complexity of the tetracyclines' pleiotropic effects has hampered the study of the actual mechanisms responsible for their immunosuppressive activity, which have remained elusive.

Tetracyclines, in particular doxycycline, are commonly used experimentally in cell lines and transgenic mice as regulators of artificial inducible/repressible transcription systems (25–27). Despite the considerable amount of evidence pointing to specific activities of tetracyclines in eukaryotic organisms, the doxycycline-regulatable systems have become widely utilized because of their purported lack of significant pleiotropic effects. We have conducted a series of control experiments to establish the effect of doxycycline treatment on our model system, i.e. in vitro activated primary mouse B lymphocytes, as a preliminary step to establishing such inducible systems for the study of B cell activation. The results we present here demonstrate that tetracyclines display several specific and dramatic inhibitory effects on B cell function in vitro at concentrations commonly achieved during antibiotic therapy and comparable to those used in inducible transgenic systems.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents and cell lines
Doxycycline (1 mg/ml in water; Sigma, St Louis, MO), chemically modified tetracyclines (CMT; 1 mg/ml in 90% ethanol, 10% DMSO; kindly provided by Collagenex Pharmaceuticals, Newton, PA), the hydroxamate KB8301 (10 mM in DMSO; PharMingen, San Diego, CA) were all stored aliquoted at –20°C. Human tissue inhibitor of metalloproteinases (TIMP)-1 and -2 (0.5 mg/ml in PBS; Chemicon International, Temecula, CA) were stored at –80°C.

In vitro activation assays
Splenocytes were derived from 4- to 16-week-old C57Bl/6 or BALB/c mice housed in specific pathogen-free conditions in the URMC Vivarium. CD19+ B cells were purified in some experiments using a Miltenyi Biotec (Bergisch-Gladbach, Germany) Midi-MACS magnetic sorting apparatus and columns with either directly conjugated anti-CD19 beads or FITC-conjugated anti-CD19 antibody and anti-FITC beads, according to the manufacturer's protocols. A single purification step consistently yielded 95–98% B220+, IgM+ cells. In some experiments, the cells were loaded prior to culture with the vital fluorescent dye 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR), which allows estimation of division cycles of proliferating cells, as described (28,29).

Splenocytes or CD19+ B cells were cultured for 5 days in complete RPMI medium, 10% FCS plus activators—either 20 µg/ml bacterial lipopolysaccharide (LPS; Sigma)/10 µg/ml dextran sulfate or 2 µg/ml anti-CD40 antibody (clone HM40-3; PharMingen)—with or without 25 ng/ml recombinant mouse IL-4 (R & D Systems, Minneapolis, MN). The cell concentration was adjusted daily to ~106/ml. All additional reagents were added from day 0 of culture at the concentrations indicated in the text. At day 5, the B cell blasts were stained for expression of surface markers and Ig heavy chain isotypes using fluorochrome-conjugated mAb (PharMingen and Southern Biotechnology Associates, Birmingham, AL) (see legends for antibody description). Stained cells were analyzed by flow cytometry on a Becton Dickinson (Mountain View, CA) FACSCalibur instrument using CellQuest software. The fraction of switched cells was quantified using CellQuest software in eight to 16 independent LPS culture experiments, depending on the isotype, and three independent experiments for anti-CD40/IL-4 cultures; specific gates were drawn according to the distribution of isotype-positive cells in inducing versus non-inducing conditions (e.g. LPS versus LPS/IL-4 for IgG3).

[3H]Thymidine incorporation was measured at 48 h of culture, using flat-bottom 96-well plates (starting concentration, 105 cells/well) with 1 µCi/well [3H]thymidine for 12–16 h.

Analysis of Ig secretion
C57Bl/6 lymphoblasts (from day 5 LPS cultures with or without doxycycline) were washed in PBS and resuspended at 106 live cells/ml in fresh culture medium with or without doxycycline. These secondary cultures were incubated for another 24 h, after which supernatants were collected and analyzed by ELISA using polyclonal anti-IgM antibodies (Southern Biotechnology Associates) on Dynatech Immulon I plates.

RNA analysis
Total RNA was extracted using the Trizol reagent (Gibco/BRL, Carlsbad, CA). Northern blot analysis was performed by electrophoresis of 10 µg of total RNA on 2% formaldehyde, 1% agarose gels, transfer onto ZetaProbe nylon membranes (BioRad, Hercules, CA) and hybridization to random-primed 32P-labeled probes (see legends for probe description). Multiprobe RNase protection assays (RPA) were performed using the mMyc probe template set with the Riboquant in vitro transcription and RPA kits (PharMingen) on RNAs from two independent experiments, with reproducible results. Amounts of 15 µg of total RNA/sample were used in each assay. The RPA products were separated by 6 M urea–PAGE and detected by autoradiography for 2–16 h.

For RT-PCR, 5 µg of total RNA from the indicated sources were reverse transcribed using random hexamer primers and Superscript reverse transcriptase (Gibco/BRL). After digestion with RNase H, the cDNA was subject to PCR using the following primers: secretory Cµ tail: 5'-CACACTGTACAATGTCTCCCT-3' and 5'-AAAATGCAACATCTCACTCTG-3'; membrane Cµ tail: 5'-TCCTCCTGAGCCTCTTCTAC-3' and 5'-CCAGACATTGCTTCAGATTG-3'. Co-amplification reactions were set up with both sets of primers, and PCR aliquots were withdrawn at 23, 25, 27, 29 and 31 cycles (1 min denaturation at 94°C, 30 s annealing at 55°C and 1 min extension at 72°C each) to control for linear amplification of both products. Amplified fragments were run on a 1.5% agarose gel and visualized by ethidium bromide staining.

Digestion–circularization PCR (DC-PCR)
Genomic DNAs were extracted from day 4 LPS B cell blasts using the Qiagen (Valencia, CA) DNeasy kit and digested to completion with the indicated restriction enzymes. The digested samples were then re-ligated in diluted conditions (360 ng/200 µl reaction) which favor re-circularization of the products, precipitated and resuspended, and 100 ng aliquots were subject to DC-PCR amplification. Published primers and conditions were used for nested PCR of EcoRI-cut and re-circularized Sµ–S{varepsilon} rearranged fragments (30) and for PCR of the non-rearranging acetylcholine receptor (AchR) gene, as an internal control (31). For Sµ–S{gamma}3 rearrangements, genomic DNAs were digested with XbaI, and the religated product subjected to a nested PCR reaction using first the primer pair 5'-GGTCAAACTTGTTACAGCCGTT-3' and 5'-ACTGAGTGTCCTCTCAACCACC-3' (30 cycles, 1 min at 94°C, 90 s at 55°C, 2 min at 72°C), followed by the primer pair 5'-ATGCACAGAGGGAAGGAAGAAG-3' and 5'-CCAGCATGTTCAACCGAAATAA-3' (30 cycles, 1 min at 94°C, 1 min at 58°C, 90 s at 72°C). Internal control for this reaction was a DC-PCR assay for the Delta-like 3 Notch ligand gene (GenBank accession no. AF068865), using the primers 5'-ATAACGTGTTTGTGGAAGTTAGAGGA-3' and 5'-CGATGATAGAGAAGGGACAAGATAGA-3'. All products were separated on a 1.5% agarose gel and visualized by ethidium bromide.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Doxycycline significantly inhibits Ig secretion and class switching in vitro
Primary mouse splenocytes were cultured in medium containing LPS/dextran, with or without the addition of IL-4, in the presence of increasing concentrations of doxycycline (in the therapeutically effective range of 1.25–5 µg/ml). At day 5, the activated B cell blasts were harvested and analyzed for expression of Ig isotypes by flow cytometry.

Treatment with 5 µg/ml doxycycline resulted in a significant decrease in the fraction of lymphoblasts positive for secondary isotypes (Fig. 1Go). On average, a 10-fold decrease was observed for IgG3 expression, 2-fold for IgG1 and 4-fold for IgG2b. Similarly, surface staining for IgE in LPS/IL-4-treated B cells was also significantly diminished (14% of normal MFI). Note, however, that this decrease, unlike that in IgG isotype-positive cells, indicates a lower level of IgE in the supernatant, rather than lower numbers of IgE-switched cells, because of passive IgE binding to the low-affinity Fc{varepsilon}RI, CD23.



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 1. Doxycycline inhibits Ig class switching in LPS-activated B cells. Day 5 lymphoblasts from LPS/dextran cultures, with (right) or without (left) addition of IL-4, were stained with antibodies to B220 (vertical axis) and Ig isotypes (horizontal axis, as indicated) and analyzed by flow cytometry. The following rat-anti-mouse mAb were used: CyChrome–anti-B220 (clone RA3-6B2), PE–anti-IgM (1B4B1), FITC–anti-IgG1 (A85-1), FITC–anti-IgG3 (R40-82), PE–anti-IgE (23G3) and PE–anti-IgG2b (LO-MG2b). Plots show the profile of 104 live lymphoblasts gated on forward/side scatter. The top row of panels shows the class switch profile of normal cultures and the bottom row shows the profile of cultures supplemented with 5 µg/ml doxycycline. The percentage of cells in the indicated gates versus total live lymphoblasts is shown. Note the marked decrease in the number of IgG3, IgG2b, IgG1 and IgMlow cells in doxycycline-treated cultures. As discussed in the text, IgE staining corresponds to the amount of passively absorbed, CD23-bound IgE. Therefore, a generalized decrease in stain intensity (measured as MFI), as opposed to lower numbers of positive cells, is observed in these samples.

 
In agreement with a block of class switching in doxycycline-treated cells, lower numbers of surface IgMdull lymphoblasts (which include isotype-switched cells) were observed (40% of normal), while IgMbright cells were correspondingly increased (Fig. 1Go). Furthermore, splenocytes activated with anti-CD40 and IL-4 displayed a similar block in expression of IgG1 (4-fold) and IgE (6.3% of normal MFI) in the presence of doxycycline (Fig. 2Go); thus, the inhibitory mechanism is not LPS specific. Class switch inhibition by doxycycline was found to be dose dependent, with a small but detectable effect observed in most cases at concentrations as low as 1.25 µg/ml doxycycline (Fig. 2Go).



View larger version (43K):
[in this window]
[in a new window]
  		Fig. 2. Doxycycline inhibits Ig class switching in CD40-activated B cells. Day 5 lymphoblasts from anti-CD40/IL-4 cultures were stained with antibodies to B220 (vertical axis) and Ig isotypes (horizontal axis, as indicated), and analyzed by flow cytometry as described in Fig. 1Go. Top, IgG1 and IgE switch profile of control cultures. Underneath, class switch profiles of cultures treated with progressively higher concentrations of doxycycline (indicated on the right). Note the dose-dependent decrease in both the fraction of IgG1+ cells and the stain intensity for IgE. A similar dose dependence was observed also in LPS cultures (not shown).

 
Secondary isotype expression was also affected in doxycycline-treated magnetically sorted CD19+ B lymphocytes (>95% B220+, IgM+ cells) and in splenic B cells from TCR ß{delta}–/– T cell-deficient mice, strongly suggesting a B cell-intrinsic mechanism for the inhibition (not shown).

As a second parameter in B cell activation, we analyzed the ability of LPS-activated lymphoblasts to secrete Ig. Day 5-activated B cells from control and doxycycline-treated cultures were washed and cultured at 106 cells/ml for a further 24 h in fresh medium. ELISA on the supernatants showed that doxycycline-treated cells secreted ~10-fold less IgM than control cells (Fig. 3AGo). Since surface IgM+ cells are actually more abundant in doxycycline cultures because of the block in class switching, the difference on a cell basis is likely even more pronounced.



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 3. Doxycycline inhibits IgM secretion by activated B cell blasts. Doxycycline-treated and control day 5 LPS lymphoblasts were plated at 106 cells/ml in fresh medium (supplemented with 5 µg/ml doxycycline when appropriate), and IgM secretion was measured 24 h later by ELISA on the culture supernatants. In three independent experiments with activated B cell blasts a 5- to 10-fold suppression in IgM production is observed in doxycycline-treated cells. The detection limit in these experiments was 20 ng/ml (dotted line).

 
Doxycycline has only marginal effects on expression of activation markers and cell proliferation
Because both terminal B cell differentiation and, especially, class switching have been correlated with cell division in vitro, we assessed the ability of doxycycline to suppress cell proliferation of LPS-stimulated B cells. Both [3H]thymidine incorporation assays at day 2 of culture and direct measurement of cell division using vital CFSE staining indicated a small, variable decrease in proliferation of doxycycline-treated cells. Specifically, doxycycline suppressed thymidine incorporation in LPS cultures to 87 ± 23% of controls in seven independent experiments (Fig. 4AGo). Treated cells also generally displayed higher day 5 CFSE staining levels than controls, suggesting that they underwent a lower number of cell divisions (Fig. 4BGo). However, in several sets of experiments no significant decrease in proliferative capacity was observed. Most importantly, there is no correlation between the degree of cell proliferation and class switch inhibition. In fact, double CFSE/IgG3 staining reveals a deficiency in secondary isotype expression even in the fraction of doxycycline-treated cells that have undergone the highest number of cell divisions (Fig. 4CGo). Thus, while doxycycline can partially inhibit cell proliferation in vitro, this is not the cause of class switch suppression.



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 4. Proliferation in doxycycline-treated cultures. (A) [3H]Thymidine incorporation in representative LPS and anti-CD40/IL-4 cultures without (open bars) or with (closed bars) 5 µg/ml doxycycline. Average and standard deviation of triplicate or quadruplicate wells are shown; each set of bars represents an independent experiment. Note the partial decrease in incorporation in some of the cultures. (B) CFSE-stained cells from day 5 LPS cultures without (thick line) or with doxycycline (thin line) were analyzed by FACS as described in Fig. 1Go. Slightly higher levels of CFSE staining in doxycycline-treated cells reflect lower proliferative capacity. However, counterstain for surface IgG3 expression using a biotin-conjugated anti-IgG3 antibody (clone R40-82) and streptavidin–PE as a second-step reagent (C) shows that in doxycycline-treated cells, even among the cells that have undergone a higher number of cell divisions (which are partially reduced compared to controls), IgG3 switching is still inhibited by over 10-fold.

 
Non-specific effects on cell viability could also be ruled out, as the number of dead cells (by forward/side scatter, Trypan blue exclusion and propidium iodide staining) was not increased in doxycycline-treated cells (not shown). Normal cell function is also indicated by the observation that early activation markers are similarly up-regulated in both doxycycline-treated cells and controls (Fig. 5Go and not shown); however, the fraction of cells expressing the Syndecan-1/CD138 plasma cell marker was decreased by ~3-fold (over five experiments) (Fig. 5Go).



View larger version (53K):
[in this window]
[in a new window]
  		Fig. 5. Expression of activation and terminal differentiation markers in doxycycline-treated LPS cultures. Cells from day 5 control and doxycycline-treated LPS (top and center panels) and LPS/IL-4 (bottom panel) were harvested and stained with antibodies to B220 (same as in Fig. 1Go), and to the activation antigens Syndecan-1/CD138 (clone 281-2, PE), B7-2/CD86 (clone GL1, PE) and CD23 (clone B3B4, FITC). Flow cytometry was performed as in Fig. 1Go. Note the reduction in the B220low, Syndecan-1+, more terminally differentiated B cells following doxycycline treatment (top). B7-2 (center) and CD23 (bottom) are up-regulated normally in doxycycline-treated cells. The decrease in the B220low, B7-2low population in doxycycline-treated cultures is also likely due to lower numbers of plasmacytoid cells expressing this phenotype. Uniformly higher levels of CD23 staining (87% higher on average over five experiments) were reproducibly observed, consistent with published data on the block of shedding of this receptor from the cell surface by MP inhibitors (43,46).

 
Doxycycline inhibits class switch recombination
Class switch recombination is blocked by mutation of the activation-induced deaminase (AID) gene, either in human autosomal hyper-IgM syndrome patients or in gene-targeted mutant mice (32,33). Using Northern blot analysis, we found no significant differences in AID mRNA levels between treated and control RNA samples (Fig. 6AGo), ruling out an effect of doxycycline on AID gene transcription.



View larger version (46K):
[in this window]
[in a new window]
  		Fig. 6. Analysis of gene expression in doxycycline-treated LPS cultures. Total RNAs from day 2 and 5 control and doxycycline-treated lymphoblasts were analyzed by Northern blotting for expression of class switch-related (A) and terminal differentiation-related (B) transcripts. The blots shown are representative of three independent experiments (two for J chain hybridization). (A) The Northern blot was sequentially hybridized with probes (indicated on the left) for the AID gene (0.7 kb cDNA fragment with the entire coding sequence), the I{gamma}2b germline transcript exon (0.5 kb HindIII–XhoI fragment), the C{gamma}2b gene (0.5 kb SacI fragment) and the 28S ribosomal RNA component (human 1.3 kb BamHI fragment). Intensity of the bands was quantified by analysis of Phosphorimager scans. Relative intensity values shown at the bottom represent the ratios of the intensity of individual bands versus the 28S loading control band, normalized to the day 2 LPS alone control RNA (set to 1). Levels of AID and I{gamma}2b transcripts are essentially unchanged by doxycycline treatment, while C{gamma}2b levels are decreased by~2- to 5-fold, depending on the experiment (mem- and sec- denote transcripts bearing the alternatively spliced membrane or secretory mRNA poly-A tail). Note that the bands for the I{gamma}2b–C{gamma}2b and VDJ–C{gamma}2b transcripts detected by the C{gamma}2b probe are partially overlapping (see the clear doublet for the secretory form of both bands, sec-C{gamma}2b/sec-I{gamma}2b), so the difference between samples may be somewhat under-estimated. (B) A Northern blot with the same samples as in (A) was hybridized with probes for the Cµ gene (0.8 kb XbaI–BamHI genomic fragment), Blimp-1 (5.6 kb XhoI fragment, complete cDNA), c-myc (0.46 kb PstI fragment), J chain (1.3 kb XbaI fragment) and 28S. Relative hybridization signals were quantified as in (A). Note the lack of up-regulation of Blimp-1 and J chain mRNA expression in doxycycline cultures, and the relatively unchanged levels of µ transcripts (60–80% of normal over four experiments). c-myc levels, which are decreased by ~2-fold between day 2 and day 5 of normal culture, were slightly increased by doxycycline treatment. (C) RNase protection assay of total RNAs from day 2 and 5 control and doxycycline-treated lymphoblasts. The PharMingen Mmyc template set, which generates probes for several mouse myc-related genes, was used. Only some indicative bands are shown: note the slight increase in c-myc mRNA levels (as already shown by Northern in A) and the lower levels of mad-4 in doxycycline-treated cells. Levels of other myc-related genes (sin-3, max and mad) are relatively unchanged.

 
Also required for class switching is the induction of germline transcripts from the heavy chain constant region genes targeted for recombination (reviewed in 34). Northern blot analysis showed, however, similar levels of steady-state levels of germline transcripts in doxycycline-treated and control cultures (Fig. 6AGo), suggesting that doxycycline inhibits a later stage in the class switch process. We therefore used a DC-PCR assay to establish whether actual switch recombination takes place in doxycycline-treated B lymphocytes. In this assay, genomic DNA fragments spanning rearranged switch regions are generated by restriction enzyme digestion and recircularized by ligation in diluted conditions. The junction of the circularized fragments can be amplified by PCR using primers mapping 5' of the Sµ region and 3' of the target S region (31); because these primers would map on different circularized fragments on unrearranged DNA, the amplification is specific to switched cells. DC-PCR analysis shows a >20-fold decrease in the amount of Sµ–S{gamma}3 rearranged fragments in doxycycline-treated cells (Fig. 7Go). Similar results were obtained for Sµ–S{varepsilon} DC-PCR assays (not shown). Together with the germline transcript expression data, these findings strongly suggest that the switch block in doxycycline-treated cells occurs at, or immediately before, the recombination stage.



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 7. Doxycycline inhibits class switching at the recombination stage. Genomic DNAs from control or doxycycline-treated day 4 LPS activated B cell blasts were digested with XbaI and recircularized. DC-PCR reactions for the Sµ–S{gamma}3 rearranged fragments (top panel) were performed on serial dilutions of religated product. The amount of substrate DNA was normalized with parallel DC-PCR amplification of the non-rearranging Delta-like ligand 3 (Dll3) gene (bottom panel). The size of the products is indicated on the right. The amount of detectable Sµ–S{gamma}3 rearranged products is >20-fold lower in reactions from doxycycline-treated cells.

 
Doxycycline inhibits expression of genes associated with terminal B cell differentiation
Terminal B cell differentiation to Ig-secreting cells is accompanied by the induction of expression of the Blimp-1 gene, which is able to promote the differentiation of surface Ig+ cell lines into Ig secretors (35). During late B cell activation, Blimp-1 up-regulation directly represses c-myc transcription, while expression of the myc antagonist mad-4 and of the secretory component of polymeric Ig, J chain are induced (35–37). We therefore analyzed the expression of these genes using Northern blot analysis and RNase protection. In agreement with the decrease in Ig secretion, doxycycline-treated in vitro-activated B cell blasts expressed 3- to 5-fold less Blimp-1 transcripts and >10-fold less J chain transcripts compared to controls (Fig. 6BGo), and failed to up-regulate mad-4 transcription (Fig. 6CGo). A small but reproducible increase in c-myc mRNA levels (~50%) was also observed (Fig. 6B and CGo). Cµ transcript expression is also only marginally affected by doxycycline treatment in LPS cultures (60–80% of normal) (Fig. 6BGo), ruling out a major modulation of IgH locus transcription by doxycycline.

Our data suggest therefore that doxycycline may inhibit the entire terminal differentiation program, resulting in coordinate alterations of an entire set of genes and markers (Blimp-1, mad-4, J chain and Syndecan-1) characteristic of the later stages of B cell activation.

Other MP inhibitors suppress class switching in activated B cells
As discussed above, tetracyclines display several pleiotropic effects on mammalian cells, only a handful of which have been thoroughly characterized. A significant property of tetracyclines is their ability to inhibit matrix MP, by reducing mRNA expression and directly blocking MP activity (6,8). MP are ubiquitously expressed and are known to play important roles in lymphocyte development and function; therefore, we tested other known inhibitors for their activity on B cells.

First, we analyzed the effect of three tetracycline analogs lacking antibiotic activity: CMT3 and CMT8, which retain MP inhibitory activity, and CMT5, which does not (reviewed in 10). Of the three reagents, CMT3 and CMT8 were able to inhibit class switching in vitro, at concentrations even lower than doxycycline (1.25 and 2.5 µg/ml respectively); CMT5, on the other hand, did not display any effect at concentrations up to 7.5 µg/ml (Fig. 8AGo).



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 8. Effects of other MP inhibitors on Ig class switching. Day 5 lymphoblasts from LPS/dextran cultures were stained with antibodies to B220 (vertical axis) IgM and IgG3 (horizontal axis, as indicated) and analyzed by flow cytometry as described in Fig. 1Go. Results are representative of three to five experiments. (A) Top, control culture; bottom, cultures treated with non-antibiotic chemically modified tetracyclines CMT3 (1.25 µg/ml), CMT5 (7.5 µg/ml) and CMT8 (2.5 µg/ml). CMT3 and CMT8, which retain MP inhibitory activity, are able to suppress class switching (lower numbers of IgG3+ and IgMlow cells), while CMT5, which has lost this activity, has no effect. (B) Top, control culture; center, doxycycline-treated culture; bottom, KB8301-treated culture. The hydroxamate KB8301 inhibits class switching to an extent comparable to that of doxycycline.

 
We then analyzed some tetracycline-unrelated MP inhibitors: TIMP-1 and -2, and the hydroxamate KB8301. Note that TIMP are rather selective inhibitors, each acting on a restricted subset of MP, while both tetracyclines and hydroxamates have a wider spectrum of activity.

Normal expression of secondary isotypes was observed in cultures treated with the endogenous inhibitors TIMP-1 and -2 (not shown), but KB8301 at a concentration of 20 µM inhibited class switching to an extent comparable to 5 µg/ml doxycycline (Fig. 8BGo). However, at this concentration no decrease in Ig secretion was observed in KB8301-treated cells (not shown).

Because tetracyclines (excluding, notably, CMT5) and hydroxamates can chelate divalent cations, including Zn2+ and Ca2+, the block in class switching could potentially be due to non-specific interference with calcium-mediated signaling. To rule out this possibility, we supplemented LPS cultures with specific calcium chelators (0.5 mM EGTA or 2.5 µM BAPTA-AM, both the highest possible non-cytotoxic concentrations in these cultures); no effect on class switching was observed (not shown).

Thus, the class switch inhibitory effect of doxycycline is common to other tetracyclines MP inhibitory activity and to at least one chemically unrelated MP inhibitor, the hydroxamate KB8301.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Following the discovery of a striking variety of non-antibiotic properties of tetracyclines, the therapeutic indications for these drugs have been successfully extended in the past 15 years to rheumatoid arthritis and periodontal disease, and potential new applications are being explored for conditions as diverse as stroke, aortic aneurysm, endotoxic shock and metastatic cancer (reviewed in 7,9,10). This is certainly in large part due to the tetracyclines' ability to inhibit the release of pro-inflammatory mediators, including NO, TNF-{alpha} and IL-1 as well as the production and activity of enzymes directly involved in tissue destruction, such as the matrix MP collagenase and stromelysin (reviewed in 7,9,10).

Evidence of more subtle but significant effects of tetracyclines on immune function is also well established, although less clearly characterized. In this report, we show that doxycycline can have specific, direct effects on B lymphocytes, selectively blocking some of the activities characteristic of activated lymphoblasts and terminally differentiated B cells. This novel finding provides a key for interpretation of some of the activities of tetracyclines on the immune system.

While doxycycline-treated B cells have on average slightly lower proliferative capacity than controls, they are able to up-regulate specific activation markers, and to express AID and heavy chain germline transcripts. These observations indicate that early activation events are largely unaffected by doxycycline treatment.

Unlike the events occurring during early B cell activation, terminal B cell differentiation is strikingly affected by doxycycline, with significant decreases in Ig secretion, J chain mRNA levels and Syndecan-1 expression (three key plasma cell markers). The entire differentiation program appears to be targeted, since doxycycline treatment also decreases expression of the Blimp-1 gene, considered a master transcription factor for terminal B cell differentiation (35), and of mad-4, also induced upon terminal differentiation (37). Consistently, c-myc expression levels, which are normally down-regulated in terminally differentiated B cells by direct Blimp-1-mediated repression (36), are slightly (51 ± 11% over three experiments) but reproducibly higher in the presence of doxycycline.

In contrast to terminal B cell differentiation, inhibition of class switching appears to be affected at the recombination stage and is not accompanied by inhibition of expression of either germline transcripts or the AID gene. Whether this is a direct effect on the recombination mechanism or indirectly mediated through some unknown intermediate factor remains to be investigated. Both in vitro class switching and terminal B cell differentiation are known to be correlated with the number of cell divisions (28,29,38), suggesting perhaps that, although largely independent phenomena, they may share some early common induction pathway. In any case, our data show that the small, variable effect of doxycycline on proliferation is most likely not responsible for the observed effects.

The ability of other MP inhibitors to mimic doxycycline's effect on class switching is particularly interesting and may suggest a previously unrecognized role of these enzymes in B cell activation. MP have already been implicated in many aspects of lymphocyte development and function: among their substrates are several members of the TNF/TNF receptor family, CD43, CD23, L-selectin (12,39–47, reviewed in 48). MP are also involved in the processing of Notch family receptors and their ligands (review in 49), and are generally expressed in a specific and regulated fashion in B and T cells (50–52). Indeed, it has already been reported that batimastat, another hydroxamate, is able to inhibit IgE, but not IgG, production in activated human and mouse B cells (43,46). In that case, the effect was correlated with the ability of this compound to inhibit the shedding of CD23, thereby amplifying a well-characterized negative feed-back loop on IgE secretion (53). Although we observe a similar effect of doxycycline on CD23 expression in LPS/IL-4-activated B cells, this mechanism cannot fully explain our observations, since in our hands other isotypes besides IgE are affected, and because we showed that doxycycline can directly act at the class switch recombination level. It is possible that differences in the specificity of batimastat, KB8301 and tetracyclines for individual MP, or in their bioavailability (unlike doxycycline, hydroxamates are highly insoluble), may be responsible for the variability in their effect on different aspects of B cell activation. Further investigation of these discrepancies may reveal whether a single or multiple pathways are involved in the inhibition of class switching and Ig secretion by these compounds.

Among the most appealing candidates for our observations are members of the TNF/TNF receptor family, including TNF-{alpha} itself, Blys/BAFF/zTNF4/Tall-1/THANK and its receptors TACI and BCMA, as well as CD30–CD30 ligand, which have all been shown to profoundly affect B cell function in vivo and in vitro (47,54–60, reviewed in 61,62). However, while these ligand–receptor pairs are generally thought to act in trans, either by secretion or cell–cell contact, our results suggest a B cell-intrinsic mechanism for the activity of MP inhibitors. Specific experiments will be required to establish whether homotypic interactions between activated B cells or autocrine stimulation through any of these pathways plays a role in the effect of MP inhibitors on class switching.

The MP-inhibitory activity of tetracyclines and hydroxamates depends at least in part on their ability to interfere with the Zn2+ ion in the MP active site (reviewed in 10,63). Indeed, loss of the ability to chelate divalent cations, such as in CMT-5, abrogates the tetracyclines MP-inhibitory activity (10,63). Because neither EGTA nor BAPTA-AM have any effect on class switching in LPS cultures, non-specific effects on Ca2+-mediated signals can be ruled out. It is possible, however, that tetracyclines and hydroxamates may also interfere with the activity of other metalloproteins, such as zinc-finger transcription factors, DNA repair enzymes or histone deacetylase (64). Interestingly, the cytidine deaminase activity of AID also has been shown to be Zn2+ dependent (65). Although the exact role of AID and cytidine deamination in class switching is still unknown, the possibility of inhibition of AID function by doxycycline is particularly intriguing.

Taken together, our observations suggest that the immunosuppressive effects of tetracyclines on humoral immune function in vivo, as judged by both reduction of basal total Ig and rheumatoid factor levels in rheumatoid arthritis patients as well as decreased responses in immunized mice, are at least in part due to a direct effect on B lymphocytes.

Another important implication of these studies concerns the use of doxycycline-inducible transcription systems for in vitro and in vivo transgenic studies. In general, it is assumed (and advertised) that doxycycline is devoid of pleiotropic effects. Yet, in vitro systems using the tetracycline-dependent activator rtTA require doxycycline concentrations up to 1 µg/ml (26), very close to the effective dose observed in our experiments. In addition, in vivo transgenic experiments are performed feeding the mice water supplemented with as much as 200 µg/ml doxycycline (27). Assuming a similar pharmacokinetics of doxycycline in mice and humans, this dose is actually higher than what normally given to patients, whose serum doxycycline concentration can rise to >8 µg/ml during treatment (66). The possibility of experimental artifacts in such systems is further amplified by the potential synergism between the multiple activities of doxycycline (even at sub-optimal concentrations) and other experimental variables. The extensive published data on the pleiotropic effects of doxycycline, exemplified by the present report, should certainly serve as a cautionary note regarding the application of these assays to complex experimental systems and the interpretation of their results.

In summary, we have described new and significant effects of doxycycline and other MP inhibitors on in vitro B cell activation. These findings provide a likely explanation for some of the observed effects of tetracyclines on humoral responses and autoimmune parameters in vivo. Detailed biochemical and molecular analyses of the activity of these compounds and of the potential role of MP in B cell activation will contribute new insights into the mechanisms of Ig secretion and class switching, and may lead to the development of new therapeutic strategies for autoimmune and allergic diseases.


   Acknowledgments
 
We wish to thank Chris Ritchlin and Sally Haas-Smith (Immunology Unit, URMC) for useful discussion of our data, sharing of reagents and technical help, and Collagenex Inc. for providing us with CMT samples. We are also grateful for the encouragement and suggestions of Bob Greenwald (Long Island Jewish Medical Center), Brad Zerler (Collagenex Inc.) and several other participants to the 1999 Gordon Conference on `Non-antibiotic Properties of Tetracyclines', and to Bortolo Nardini for inspiration. This work was funded in part by NIH grants RO1AI45012 to A .B., RO1AI37123 and RO1HD36293 to R. A. I., and R29CA73696 to F. M. Y.; I. K. was supported by training grant AI07169 and S. L. by training grant AI07285. This paper is dedicated to Dongming's memory.


   Abbreviations
 
CFSE 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester
CMT chemically modified tetracycline
DC-PCR digestion-circularization PCR
LPS lipopolysaccharide
MP metalloproteinase
RPA RNase protection assay
TIMP tissue inhibitor of metalloproteinases
TNF tumor necrosis factor

   Notes
 
Transmitting editor: C. J. Paige

Received 8 December 2000, accepted 6 April 2001.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Amin, A. R., Attur, M. G., Thakker, G. D., Patel, P. D., Vyas, P. R., Patel, R. N., Patel, I. R. and Abramson, S. B. 1996. A novel mechanism of action of tetracyclines: effects on nitric oxide synthases. Proc. Natl Acad. Sci. USA 93:14014.[Abstract/Free Full Text]
  2. Amin, A. R., Patel, R. N., Thakker, G. D., Lowenstein, C. J., Attur, M. G. and Abramson, S. B. 1997. Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by doxycycline and chemically modified tetracyclines. FEBS Lett. 410:259.[Web of Science][Medline]
  3. Pruzanski, W., Greenwald, R. A., Street, I. P., Laliberte, F., Stefanski, E. and Vadas, P. 1992. Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline. Biochem. Pharmacol. 44:1165.[Web of Science][Medline]
  4. Patel, R. N., Attur, M. G., Dave, M. N., Patel, I. V., Stuchin, S. A., Abramson, S. B. and Amin, A. R. 1999. A novel mechanism of action of chemically modified tetracyclines: inhibition of COX-2-mediated prostaglandin E2 production. J. Immunol. 163:3459.[Abstract/Free Full Text]
  5. Attur, M. G., Patel, R. N., Patel, P. D., Abramson, S. B. and Amin, A. R. 1999. Tetracycline up-regulates COX-2 expression and prostaglandin E2 production independent of its effect on nitric oxide. J. Immunol. 162:3160.[Abstract/Free Full Text]
  6. Greenwald, R. A., Golub, L. M., Lavietes, B., Ramamurthy, N. S., Gruber, B., Laskin, R. S. and McNamara, T. F. 1987. Tetracyclines inhibit human synovial collagenase in vivo and in vitro. J. Rheumatol. 14:28.[Web of Science][Medline]
  7. Golub, L. M., Ramamurthy, N. S., McNamara, T. F., Greenwald, R. A. and Rifkin, B. R. 1991. Tetracyclines inhibit connective tissue breakdown: new therapeutic implications for an old family of drugs. Crit. Rev. Oral Biol. Med. 2:297.[Abstract/Free Full Text]
  8. Hanemaaijer, R., Visser, H., Koolwijk, P., Sorsa, T., Salo, T., Golub, L. M. and van Hinsbergh, V. W. 1998. Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells. Adv. Dental Res. 12:114.[Abstract/Free Full Text]
  9. Vernillo, A. T. and Rifkin, B. R. 1998. Effects of tetracyclines on bone metabolism. Adv. Dental Res. 12:56.[Abstract/Free Full Text]
  10. Golub, L. M., Lee, H. M., Ryan, M. E., Giannobile, W. V., Payne, J. and Sorsa, T. 1998. Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Adv. Dental Res. 12:12.[Abstract/Free Full Text]
  11. Shapira, L., Soskolne, W. A., Houri, Y., Barak, V., Halabi, A. and Stabholz, A. 1996. Protection against endotoxic shock and lipopolysaccharide-induced local inflammation by tetracycline: correlation with inhibition of cytokine secretion. Infect. Immun. 64:825.[Abstract/Free Full Text]
  12. Liu, J., Kuszynski, C. A. and Baxter, B. T. 1999. Doxycycline induces Fas/Fas ligand-mediated apoptosis in Jurkat T lymphocytes. Biochem. Biophys. Res. Commun. 260:562.[Web of Science][Medline]
  13. Tilley, B. C., Alarcon, G. S., Heyse, S. P., Trentham, D. E., Neuner, R., Kaplan, D. A., Clegg, D. O., Leisen, J. C., Buckley, L., Cooper, S. M., et al. 1995. Minocycline in rheumatoid arthritis. A 48-week, double-blind, placebo-controlled trial. MIRA Trial Group. Ann. Intern. Med. 122:81.[Abstract/Free Full Text]
  14. Kloppenburg, M., Dijkmans, B. A., Verweij, C. L. and Breedveld, F. C. 1996. Inflammatory and immunological parameters of disease activity in rheumatoid arthritis patients treated with minocycline. Immunopharmacology 31:163.[Web of Science][Medline]
  15. Van den Bogert, C. and Kroon, A. M. 1982. Effects of oxytetracycline on in vivo proliferation and differentiation of erythroid and lymphoid cells in the rat. Clin. Exp. Immunol. 50:327.[Web of Science][Medline]
  16. Bellahsene, A. and Forsgren, A. 1985. Effect of doxycycline on immune response in mice. Infect. Immun. 48:556.[Abstract/Free Full Text]
  17. Woo, P. C., Tsoi, H. W., Wong, L. P., Leung, H. C. and Yuen, K. Y. 1999. Antibiotics modulate vaccine-induced humoral immune response. Clin. Diagn. Lab. Immunol. 6:832.[Abstract/Free Full Text]
  18. Thong, Y. H. and Ferrante, A. 1979. Inhibition of mitogen-induced human lymphocyte proliferative responses by tetracycline analogues. Clin. Exp. Immunol. 35:443.[Web of Science][Medline]
  19. Potts, R. C., Hassan, H. A., Brown, R. A., MacConnachie, A., Gibbs, J. H., Robertson, A. J. and Beck, J. S. 1983. In vitro effects of doxycycline and tetracycline on mitogen stimulated lymphocyte growth. Clin. Exp. Immunol. 53:458.[Web of Science][Medline]
  20. Ibrahim, M. S., Maged, Z. A., Haron, A., Khalil, R. Y. and Attallah, A. M. 1988. Antibiotics and immunity: effects of antibiotics on mitogen responsiveness of lymphocytes and interleukin-2 production. Chemioterapia 7:369.[Web of Science][Medline]
  21. Kloppenburg, M., Verweij, C. L., Miltenburg, A. M., Verhoeven, A. J., Daha, M. R., Dijkmans, B. A. and Breedveld, F. C. 1995. The influence of tetracyclines on T cell activation. Clin. Exp. Immunol. 102:635.[Web of Science][Medline]
  22. Preus, H., Tollefsen, T. and Morland, B. 1987. Effects of tetracycline on human monocyte phagocytosis and lymphocyte proliferation. Acta Odontol. Scand. 45:297.[Web of Science][Medline]
  23. Ingham, E., Turnbull, L. and Kearney, J. N. 1991. The effects of minocycline and tetracycline on the mitotic response of human peripheral blood-lymphocytes. J. Antimicrob. Chemother. 27:607.[Abstract/Free Full Text]
  24. Myers, M. J., Farrell, D. E. and Henderson, M. 1995. Oxy- tetracycline-mediated alteration of murine immunocompetence. Pathobiology 63:270.[Web of Science][Medline]
  25. Gossen, M. and Bujard, H. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl Acad. Sci. USA 89:5547.[Abstract/Free Full Text]
  26. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W. and Bujard, H. 1995. Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766.[Abstract/Free Full Text]
  27. Kistner, A., Gossen, M., Zimmermann, F., Jerecic, J., Ullmer, C., Lubbert, H. and Bujard, H. 1996. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc. Natl Acad. Sci. USA 93:10933.[Abstract/Free Full Text]
  28. Hodgkin, P. D., Lee, J. H. and Lyons, A. B. 1996. B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med. 184:277.[Abstract/Free Full Text]
  29. Hasbold, J., Lyons, A. B., Kehry, M. R. and Hodgkin, P. D. 1998. Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4. Eur. J. Immunol. 28:1040.[Web of Science][Medline]
  30. Xu, L. and Rothman, P. 1994. IFN-{gamma} represses {varepsilon} germline transcription and subsequently down-regulates switch recombination to {varepsilon}. Int. Immunol. 6:515.[Abstract/Free Full Text]
  31. Chu, C. C., Paul, W. E. and Max, E. E. 1992. Quantitation of immunoglobulin m-{gamma}1 heavy chain switch region recombination by a digestion-circularization polymerase chain reaction method. Proc. Natl Acad. Sci. USA 89:6978.[Abstract/Free Full Text]
  32. Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y. and Honjo, T. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553.[Web of Science][Medline]
  33. Revy, P., Muto, T., Levy, Y., Geissmann, F., Plebani, A., Sanal, O., Catalan, N., Forveille, M., Dufourcq-Labelouse, R., Gennery, A., Tezcan, I., Ersoy, F., Kayserili, H., Ugazio, A. G., Brousse, N., Muramatsu, M., Notarangelo, L. D., Kinoshita, K., Honjo, T., Fischer, A. and Durandy, A. 2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome. Cell 102:565.[Web of Science][Medline]
  34. Stavnezer, J. 1996. Immunoglobulin class switching. Curr. Opin. Immunol. 8:199.[Web of Science][Medline]
  35. Turner, C. A., Jr, Mack, D. H. and Davis, M. M. 1994. Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell 77:297.[Web of Science][Medline]
  36. Lin, Y., Wong, K. and Calame, K. 1997. Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation. Science 276:596.[Abstract/Free Full Text]
  37. Knodel, M., Kuss, A. W., Lindemann, D., Berberich, I. and Schimpl, A. 1999. Reversal of Blimp-1-mediated apoptosis by A1, a member of the Bcl-2 family. Eur. J. Immunol. 29:2988.[Web of Science][Medline]
  38. Hodgkin, P. D., Yamashita, L. C., Coffman, R. L. and Kehry, M. R. 1990. Separation of events mediating B cell proliferation and Ig production by using T cell membranes and lymphokines. J. Immunol. 145:2025.[Abstract]
  39. Bazil, V. and Strominger, J. L. 1993. CD43, the major sialoglycoprotein of human leukocytes, is proteolytically cleaved from the surface of stimulated lymphocytes and granulocytes. Proc. Natl Acad. Sci. USA 90:3792.[Abstract/Free Full Text]
  40. Kayagaki, N., Kawasaki, A., Ebata, T., Ohmoto, H., Ikeda, S., Inoue, S., Yoshino, K., Okumura, K. and Yagita, H. 1995. Metalloproteinase-mediated release of human Fas ligand. J. Exp. Med. 182:1777.[Abstract/Free Full Text]
  41. Feehan, C., Darlak, K., Kahn, J., Walcheck, B., Spatola, A. F. and Kishimoto, T. K. 1996. Shedding of the lymphocyte L-selectin adhesion molecule is inhibited by a hydroxamic acid-based protease inhibitor. Identification with an L-selectin-alkaline phosphatase reporter. J. Biol. Chem. 271:7019.[Abstract/Free Full Text]
  42. Preece, G., Murphy, G. and Ager, A. 1996. Metalloproteinase-mediated regulation of L-selectin levels on leucocytes. J. Biol. Chem. 271:11634.[Abstract/Free Full Text]
  43. Christie, G., Barton, A., Bolognese, B., Buckle, D. R., Cook, R. M., Hansbury, M. J., Harper, G. P., Marshall, L. A., McCord, M. E., Moulder, K., Murdock, P. R., Seal, S. M., Spackman, V. M., Weston, B. J. and Mayer, R. J. 1997. IgE secretion is attenuated by an inhibitor of proteolytic processing of CD23 (Fc{varepsilon}RII). Eur. J. Immunol. 27:3228.[Web of Science][Medline]
  44. Black, R. A., Rauch, C. T., Kozlosky, C. J., Peschon, J. J., Slack, J. L., Wolfson, M. F., Castner, B. J., Stocking, K. L., Reddy, P., Srinivasan, S., Nelson, N., Boiani, N., Schooley, K. A., Gerhart, M., Davis, R., Fitzner, J. N., Johnson, R. S., Paxton, R. J., March, C. J. and Cerretti, D. P. 1997. A metalloproteinase disintegrin that releases tumour-necrosis factor-{alpha} from cells. Nature 385:729.[Medline]
  45. Moss, M. L., Jin, S. L., Milla, M. E., Bickett, D. M., Burkhart, W., Carter, H. L., Chen, W. J., Clay, W. C., Didsbury, J. R., Hassler, D., Hoffman, C. R., Kost, T. A., Lambert, M. H., Leesnitzer, M. A., McCauley, P., McGeehan, G., Mitchell, J., Moyer, M., Pahel, G., Rocque, W., Overton, L. K., Schoenen, F., Seaton, T., Su, J. L., Becherer, J. D., et al. 1997. Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 385:733.[Medline]
  46. Marolewski, A. E., Buckle, D. R., Christie, G., Earnshaw, D. L., Flamberg, P. L. Marshall, L. A., Smith, D. G. and Mayer, R. J. 1998. CD23 (Fc{varepsilon}RII) release from cell membranes is mediated by a membrane-bound metalloprotease. Biochem. J. 333:573.
  47. Schneider, P., MacKay, F., Steiner, V., Hofmann, K., Bodmer, J. L., Holler, N., Ambrose, C., Lawton, P., Bixler, S., Acha-Orbea, H., Valmori, D., Romero, P., Werner-Favre, C., Zubler, R. H., Browning, J. L. and Tschopp, J. 1999. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J. Exp. Med. 189:1747.[Abstract/Free Full Text]
  48. Yamamoto, S., Higuchi, Y., Yoshiyama, K., Shimizu, E., Kataoka, M., Hijiya, N. and Matsuura, K. 1999. ADAM family proteins in the immune system. Immunol. Today 20:278.[Web of Science][Medline]
  49. Osborne, B. and Miele, L. 1999. Notch and the immune system. Immunity 11:653.[Web of Science][Medline]
  50. Johnatty, R. N., Taub, D. D., Reeder, S. P., Turcovski-Corrales, S. M., Cottam, D. W., Stephenson, T. J. and Rees, R. C. 1997. Cytokine and chemokine regulation of proMMP-9 and TIMP-1 production by human peripheral blood lymphocytes. J. Immunol. 158:2327.[Abstract]
  51. Stetler-Stevenson, M., Mansoor, A., Lim, M., Fukushima, P., Kehrl, J., Marti, G., Ptaszynski, K., Wang, J. and Stetler-Stevenson, W. G. 1997. Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in reactive and neoplastic lymphoid cells. Blood 89:1708.[Abstract/Free Full Text]
  52. Di Girolamo, N., Tedla, N., Lloyd, A. and Wakefield, D. 1998. Expression of matrix metalloproteinases by human plasma cells and B lymphocytes. Eur. J. Immunol. 28:1773.[Web of Science][Medline]
  53. Luo, H., Hofstetter, H., Banchereau, J. and Delespesse, G. 1991. Cross-linking of CD23 antigen by its natural ligand (IgE) or by anti-CD23 antibody prevents B lymphocyte proliferation and differentiation. J. Immunol. 146:2122.[Abstract]
  54. Shanebeck, K. D., Maliszewski, C. R., Kennedy, M. K., Picha, K. S., Smith, C. A., Goodwin, R. G. and Grabstein, K. H. 1995. Regulation of murine B cell growth and differentiation by CD30 ligand. Eur. J. Immunol. 25:2147.[Web of Science][Medline]
  55. Cerutti, A., Schaffer, A., Shah, S., Zan, H., Liou, H. C., Goodwin, R. G. and Casali, P. 1998. CD30 is a CD40-inducible molecule that negatively regulates CD40-mediated immunoglobulin class switching in non-antigen-selected human B cells. Immunity 9:247.[Web of Science][Medline]
  56. Cerutti, A., Schaffer, A., Goodwin, R. G., Shah, S., Zan, H., Ely, S. and Casali, P. 2000. Engagement of CD153 (CD30 ligand) by CD30+ T cells inhibits class switch DNA recombination and antibody production in human IgD+ IgM+ B cells. J. Immunol. 165:786.[Abstract/Free Full Text]
  57. Moore, P. A., Belvedere, O., Orr, A., Pieri, K., LaFleur, D. W., Feng, P., Soppet, D., Charters, M., Gentz, R., Parmelee, D., Li, Y., Galperina, O., Giri, J., Roschke, V., Nardelli, B., Carrell, J., Sosnovtseva, S., Greenfield, W., Ruben, S. M., Olsen, H. S., Fikes, J. and Hilbert, D. M. 1999. BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285:260.[Abstract/Free Full Text]
  58. Mukhopadhyay, A., Ni, J., Zhai, Y., Yu, G. L. and Aggarwal, B. B. 1999. Identification and characterization of a novel cytokine, THANK, a TNF homologue that activates apoptosis, nuclear factor-kappaB, and c-Jun NH2-terminal kinase. J. Biol. Chem. 274:15978.[Abstract/Free Full Text]
  59. Shu, H. B., Hu, W. H. and Johnson, H. 1999. TALL-1 is a novel member of the TNF family that is down-regulated by mitogens. J. Leuk. Biol. 65:680.[Abstract]
  60. Gross, J. A., Johnston, J., Mudri, S., Enselman, R., Dillon, S. R., Madden, K., Xu, W., Parrish-Novak, J., Foster, D., Lofton-Day, C., Moore, M., Littau, A., Grossman, A., Haugen, H., Foley, K., Blumberg, H., Harrison, K., Kindsvogel, W. and Clegg, C. H. 2000. TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature404:995.[Medline]
  61. Kwon, B., Youn, B. S. and Kwon, B. S. 1999. Functions of newly identified members of the tumor necrosis factor receptor/ligand superfamilies in lymphocytes. Curr. Opin. Immunol. 11:340.[Web of Science][Medline]
  62. Gravestein, L. A. and Borst, J. 1998. Tumor necrosis factor receptor family members in the immune system. Semin. Immunol. 10:423.[Web of Science][Medline]
  63. Kleiner, D. E. J. and Stetler-Stevenson, W. G. 1993. Structural biochemistry and activation of matrix metalloproteases. Curr. Opin. Cell Biol. 5:891.[Medline]
  64. Finnin, M. S., Donigian, J. R., Cohen, A., Richon, V. M., Rifkind, R. A., Marks, P. A. Breslow, R. and Pavletich, N. P. 1999. Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature 401:188.[Medline]
  65. Muramatsu, M., Sankaranand, V. S., Anant, S., Sugai, M., Kinoshita, K., Davidson, N. O. and Honjo, T. 1999. Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J. Biol. Chem. 274:18470.[Abstract/Free Full Text]
  66. Karlsson, M., Hammers, S., Nilsson-Ehle, I., Malmborg, A. S. and Wretlind, B. 1996. Concentrations of doxycycline and penicillin G in sera and cerebrospinal fluid of patients treated for neuroborreliosis. Antimicrob. Agents Chemother. 40:1104.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Br J OphthalmolHome page
V A Smith, D Khan-Lim, L Anderson, S D Cook, and A D Dick
Does orally administered doxycycline reach the tear film?
Br J Ophthalmol, June 1, 2008; 92(6): 856 - 859.
[Abstract] [Full Text] [PDF]

Home page
 Br J OphthalmolHome page
V A Smith and S D Cook
Doxycycline--a role in ocular surface repair
Br J Ophthalmol, May 1, 2004; 88(5): 619 - 625.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Peitz, K. Pfannkuche, K. Rajewsky, and F. Edenhofer
Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: A tool for efficient genetic engineering of mammalian genomes
PNAS, April 2, 2002; 99(7): 4489 - 4494.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Peitz, K. Pfannkuche, K. Rajewsky, and F. Edenhofer
Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: A tool for efficient genetic engineering of mammalian genomes
PNAS, April 2, 2002; 99(7): 4489 - 4494.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Kuzin, I. I.
Right arrow Articles by Bottaro, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuzin, I. I.
Right arrow Articles by Bottaro, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?