| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
CUTTING EDGE |
5: A Paradigm Revisited1
Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus-Fiebiger Center, University of Erlangen-Nürnberg, Erlangen, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsEffect of an incomplete...{micro}HC induces cell growth...Sp6-{micro}HC is transported to...DiscussionReferences |
|---|
5. In 5-/- mice, pro-B cell maturation is impaired, but not completely blocked, implying that a µHC induces differentiation signals in the absence of 5. Using a mouse model, in which transgenic µHC expression can be controlled by tetracycline, we show that in the absence of 5, the transgenic µHC promotes in vivo differentiation of pro-B cells, induces IL-7-dependent cell growth, and is expressed on the surface of pre-B cells. Our findings not only show that an incomplete pre-BCR can initiate signals, but also challenge the paradigm that an IgHC must associate with an IgLC or a SLC to gain transport and signaling competency. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsEffect of an incomplete...{micro}HC induces cell growth...Sp6-{micro}HC is transported to...DiscussionReferences |
|---|
5 (6), as well as with the signal module Ig
/
into a signal-competent pre-B cell receptor (pre-BCR). Pre-BCR-positive cells divide four to six times (7, 8) and develop into small non-cycling pre-B cells and subsequently into surface IgM-positive B cells.
The assembly of a complete pre-BCR seems to be a prerequisite for inducing proliferative expansion of pre-B cells, since no, or only a few, pre-B cells are detectable in the bone marrow (BM) of mice with mutated (6, 9) or deleted pre-BCR components (2, 3, 4, 5, 10, 11). Further, the frequency of cycling cells is increased in the pre-BCR-positive BM population (12). Most importantly, tetracycline-controlled de novo induction of a µHC initiates stroma cell-dependent in vitro growth of CD19+ BM cells from a mouse able to synthesize a complete pre-BCR, but not of CD19+ BM cells from a 5- mouse (13).
In contrast, a µHC alone seems to be sufficient to mediate allelic exclusion and induce differentiation of some pro-B cells (reviewed in Refs.3 and 5), since allelically excluded IgM-positive B cells are present at a lower frequency in peripheral lymphatic organs of 5- (14) and other SLC- mice (reviewed in Ref.5). One explanation could be that some µHC+ pre-B cells are rescued by a precocious IgL rearrangement (14, 15). Alternatively, some µHCs might be transported in the absence of a complete SLC to the cell surface and induce some survival and/or proliferative signals, thereby giving a pre-B cell enough time to rearrange its IgL locus and develop into an IgM-positive B cell.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsEffect of an incomplete...{micro}HC induces cell growth...Sp6-{micro}HC is transported to...DiscussionReferences |
|---|
Mice were maintained under specific pathogen-free conditions in the animal unit of the Nikolaus-Fiebiger Center (Erlangen, Germany) and genotyped by PCR and Southern blotting.
Culturing and counting of pro-B cells
Pro-B cells were isolated from BM of 4- to 6-wk-old mice with magnetic CD19-beads (Miltenyi Biotec, Cologne, Germany) and cultured as described (13) with and without tetracycline (100 ng/ml), recombinant murine IL-7 (BD Biosciences, San Jose, CA), or 2% supernatant from IL-7-transfected J558L cells (a gift from Dr. F. Melchers, Basel Institute of Immunology, Basel, Switzerland). Cell counts of live cells as defined by forward- and side-scatter were determined by flow cytometry as described (13).
Antibodies
Rat mAbs against mouse VpreB (clone R5, Ref.16) were a gift from M. Cooper (University of Alabama, Birmingham, AL). mAbs against mouse 5 (clone LM34) and mouse µHC (clone B7-6) have previously been described (17). Abs were isolated on protein G-Sepharose from hybridoma supernatants. B7-6 mAb was biotinylated using standard procedures. Rat mAbs against CD19, c-kit, CD25 as well as blocking anti-mouse FcR Abs were obtained from BD PharMingen (San Diego, CA); the anti-FcR Abs were tested for their binding capacity by flow cytometry with the pre-B cell line NFS5. FITC- and Cy5-conjugated goat Abs against-mouse µHC were from Southern Biotechnology Associates (Birmingham, AL) and PE-streptavidin from Molecular Probes (Eugene, OR).
Flow cytometry
A total of 5 x 105 cells were stained with respective primary and secondary Abs as described (17) for 30 min on ice. For intracellular staining, cells were fixed and permeabilized using the Fix and Perm kit (An der Grub Biotechnologies, Kaumberg, Austria). Stained cells were examined in a FACSCalibur (BD Biosciences, San Jose, CA). Data were analyzed with the Cell Quest software (BD Biosciences). Statistics were performed with SigmaPlot software (RockWare, Golden, CO).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsEffect of an incomplete...{micro}HC induces cell growth...Sp6-{micro}HC is transported to...DiscussionReferences |
|---|
5. The model is based on the so-called double-transgenic Rag2-/- (dTg/Rag2-) mouse, in which the expression of a transgenic µHC utilizing the VH region from the Sp6 hybridoma can be controlled by tetracycline (Ref.13 and Fig. 1A). Mice that were obtained by intercrossing dTg/Rag2- with 5- mice served as a source for CD19+, µHC-inducible 5-, as well as 5+ pro-B cells, which were isolated by MACS from the BM of mice that had received tetracycline (200 µg/ml) for 7 days in the drinking water (i.e., development was arrested at the pro-B cell stage).
|
5 was detected only in 5+/CD19+ cells, whereas VpreB was present in both 5+ and 5- CD19+ cells (Fig. 1B). Lower levels of VpreB in 5- cells are very likely due to an increase in turnover of free VpreB (16, 18). In the absence of tetracycline, similar levels of cytoplasmic µHC were detected in 
50% of the cells, regardless whether 5 was present (Fig. 1C, black histograms). As expected, µHC+ cells were absent in cultures containing tetracycline (Fig. 1C, gray histograms). Three-color flow cytometry revealed that transgenic and endogenous µHCs are produced at comparable levels in B220low/CD25-pro-B cells (gated in dot plot diagrams in Fig. 1D) from dTg and wild-type C57BL/6 mice, respectively. |
Effect of an incomplete pre-BCR on B cell differentiation in vivo |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsEffect of an incomplete...{micro}HC induces cell growth...Sp6-{micro}HC is transported to...DiscussionReferences |
|---|
5+ and 5- dTg mice by flow cytometry (Fig. 2). As expected from published data (13), transgenic µHC rescued the block at the pro-B cell stage observed in Rag2- mice, since CD19+/CD25+/c-kit- pre-B cells could easily be detected in dTg/Rag2-/5+ mice (left plots), but not in single-transgenic tetracycline controllable transactivator (tTA)/Rag2-/5- mice (right plots). However, to our big surprise c-kit-/CD25+ pre-B cells, albeit at lower frequencies (three times less, Fig. 2) and numbers (two and three times less, data not shown), were also present in dTg/Rag2-/5- mice (middle plots), indicating that the transgenic µHC initiates intracellular differentiation signals, although with a lower efficiency, in the absence of 5. Since a µHC assembles, even in the absence of 5, with the signal transducer Ig/ (19, 20) and VpreB (21, 22), differentiation signals could be initiated in a 5- mouse by an incomplete pre-BCR consisting of µHC, Ig/, and VpreB.
|
|
µHC induces cell growth and reduces cell death rate in the absence of 5
|
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsEffect of an incomplete...{micro}HC induces cell growth...Sp6-{micro}HC is transported to...DiscussionReferences |
|---|
5-less pre-BCR on B cell maturation could be a consequence of an increased proliferation and/or survival rate of early µHC-positive pre-B cells, we compared the in vitro growth behavior of CD19+ BM cells from dTg/Rag2-/5+ mice to that from dTg/Rag2-/5- mice. In the presence of IL-7 (Fig. 3, upper panel), numbers of CD19+ BM cells from dTg/Rag2-/5+ mice increased in the absence of tetracycline (i.e., a complete pre-BCR is synthesized) 8-fold after 4 days in culture (•) when compared with cultures containing tetracycline, i.e., the transgenic µHC gene is off (
). Unexpectedly, µHC induction in CD19+ BM cultures from dTg/Rag2-/5- mice (
) also resulted in a reproducible (three independent experiments) and significant increase (p < 0.05) in cell numbers. However, when compared with cultures of CD19+ BM cells from 5+ mice, only a 2- to 3-fold increase in cell numbers was observed in CD19+ BM cultures from 5- mice. The quantitative difference in signaling of a wild-type pre-BCR and a 5-less pre-BCR was corroborated in IL-7 titration experiments (data not shown), which showed that IL-7 concentrations below 1 ng/ml still supported µHC-induced growth of 5+ but not of 5-negative CD19+ BM cells. Cell cycle analyses revealed that the increase in growth rate after µHC induction was at least in part due to an increase in proliferation (data not shown).
|
5- and 5+ BM cultures were very similar (see Fig. 1B for a 48 h time point). A negative effect of tetracycline as well as the tetracycline-tTA complex on pro-B cell growth could also be excluded, since growth curves of CD19+ BM cultures from Rag2- and tTA/Rag2- mice were almost identical, regardless whether tetracycline was present (Fig. 3, middle and right diagrams). Hence, a µHC initiates in an IL-7-dependent manner proliferation signals in pre-B cells in the absence of a complete SLC.
In the absence of IL-7 (Fig. 3, lower panel), numbers of µHC-induced dTg/Rag2-/5+/CD19+ BM cells initially dropped and stayed then constant over a period of three days (•, lower left diagram), indicating that a complete pre-BCR induces in the absence of IL-7 survival but no proliferation signals. In contrast, numbers of dTg/Rag2-/5-/CD19+ BM cells decreased rapidly in the absence of IL-7, regardless whether µHC was induced (squares, lower left diagram). However, the rate of cell death was slightly, but significantly (p < 0.0014) and reproducibly (three independent experiments) reduced in µHC-induced cultures () when compared with cultures containing tetracycline (
). Hence, a complete pre-BCR induces in the absence of IL-7 an efficient survival signal in in vitro cultured CD19+ BM cells; in contrast, a 5-less pre-BCR cannot prevent the death of CD19+ BM cells, but slightly prolongs the survival time of µHC+/5- BM cells when compared with µHC-/5- cells.
|
Sp6-µHC is transported to the cell surface in the absence of 5
|
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsEffect of an incomplete...{micro}HC induces cell growth...Sp6-{micro}HC is transported to...DiscussionReferences |
|---|
/ seems unlikely, since an ER-trapped µHC/Ig/ complex facilitates neither differentiation of pro-B into pre-B cells (6) nor pervanadate-induced tyrosine phosphorylation of Ig/ in a pre-B cell line (20). ER-signals through the so-called unfolded protein response (24) can also be excluded, since transgenic µHC induction in CD19+/5- BM cells did not affect levels of typical unfolded protein response components, such as XBP-1, BiP, and CHOP (data not shown).
We, therefore, entertained the possibility that, in contrast to other µHCs (6, 21, 25), the transgenic Sp6-µHC is transported to the cell surface in the absence of a complete SLC. To our big surprise, goat (data not shown) as well as rat Abs against µHC detected in six independent flow experiments (Fig. 4A) surface µHCs in the absence of tetracycline (open right histograms) on 40–65% of 5+ as well as 5- dTg/Rag2-/CD19+ BM cells. The observed frequency of surface µHC+ cells was, however, expected, since only 40–60% of the cells produced cytoplasmic µHCs (open left histograms in Fig. 4A). To verify that the asymmetric histograms for surface µHC staining (Fig. 4A, right histograms) contain indeed signals of surface µHC-positive and negative cells, µHC-induced CD19+ BM cells from 5+ and 5- dTg/Rag2- mice were first membrane-stained with Cy5-conjugated anti-µHC Abs, fixed, restained for cytoplasmic µHC with FITC-conjugated Abs, and finally analyzed by flow cytometry (Fig. 4B). When we gated for cytoplasmic µHC- (gray dots) and µHC+ cells (black dots), we found that cytoplasmic µHC+ cells showed an uniformly higher membrane fluorescence (open histograms) than cytoplasmic µHC- cells (gray histograms), regardless whether 5 was present. Hence the Sp6-µHC is transported to the cell surface in the absence of a complete SLC.
|
5 and speculate that a complex consisting of the Sp6-µHC, Ig/ and maybe VpreB initiates signals from the cell surface.
Surface µHC levels were lower on 5- than on 5+ CD19+ cells (see Fig. 4A and mean fluorescence intensities (MFI) in histograms of Fig. 4B). This was not due to lower cytoplasmic µHC levels in 5-/CD19+ BM cells, since MFI for intracellular µHC staining was even slightly increased in the absence of 5 (right histograms in Fig. 4A and MFI in middle plots of Fig. 4B). Reduced surface µHC levels on 5-/CD19+ cells could explain a µHC’s diminished ability to promote efficient pre-B cell differentiation in vivo (Fig. 2) and growth and survival in vitro (Fig. 3) in the absence of a complete SLC.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsEffect of an incomplete...{micro}HC induces cell growth...Sp6-{micro}HC is transported to...DiscussionReferences |
|---|
5 challenges the paradigm that a full-length µHC must pair with SLC or IgL to gain transport (23) and signaling competency (20). However, not all µHCs might be transported to the cell surface in the absence of a SLC. For example, several VH81X-µHCs that differ in their CDR3 regions do not associate with 5 and are, therefore, trapped in the ER (6, 21, 25). Consequently, such a µHC does not foster the maturation of pro-B into pre-B cells in a transgenic mouse (6). That VpreB alone confers transport competency to a µHC can be excluded, since small numbers of mature and allelically excluded B lymphocytes are also present in a triple-knock out mouse lacking VpreB1, VpreB2, and 5 (10). Transport competency in the absence of a complete SLC might rather be an intrinsic feature of a µHC that has the mightiness to assemble in the absence of an IgLC or SLC into a transport-competent µHC dimer with perfectly folded and paired Cµ1 and VH domains, a reaction that is usually prevented for most of µHCs due to the inability of their VH regions to homodimerize (26). Support for this idea comes from the finding that full-length human µHCs utilizing VH3–23 regions are secreted in the absence of a conventional IgLC (27).
In our system, signaling via a complete as well as a 5-less pre-BCR is initiated in the absence of stroma cells, a finding that challenges the existence of a stromal ligand-mediated pre-BCR signaling pathway (17, 28). However, IL-7 could compensate for the absence of a stroma cell-derived pre-BCR ligand in our experimental setting, since in vitro proliferation of µHC+ BM cells could only be observed in the presence of high doses of IL-7. Data from Hess et al. (13) showed that µHC+/5-/CD19+ BM cells do not grow when cultured only in the presence of ST2 stroma cells, thereby supporting the idea that in vitro proliferation of µHC+ pre-B cells is dependent either on high doses of IL-7 or a signal provided by a 5-dependent interaction of the pre-BCR with a stroma cell ligand (17, 28). The latter might be responsible for proliferative responses in an in vivo situation. However, in the absence of 5, the only signaling can come from an incomplete pre-BCR, i.e., a µHC/Ig complex, which results in weak proliferation and/or prolonged survival of a µHC+ pre-B cell, thereby giving this cell enough time to rearrange its IgL genes and initiate IgM-mediated survival signals.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Both W.S. and S.M. contributed equally to the manuscript.
3 Address correspondence and reprint requests to Dr. Hans-Martin Jäck, Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus-Fiebiger Center, University of Erlangen-Nürnberg, Glückstrasse 6, D-91054 Erlangen, Germany. E-mail address: HJAECK{at}molmed.uni-erlangen.de
4 Abbreviations used in this paper: µHC, µ H chain; SLC, surrogate L chain; pre-BCR, pre-B cell receptor; BM, bone marrow; tTA, tetracycline controllable transactivator; BiP, H chain binding protein; ER, endoplasmic reticulum; MFI, mean fluorescence intensities; dTg, double transgenic.
Received for publication June 24, 2003. Accepted for publication August 11, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsEffect of an incomplete...{micro}HC induces cell growth...Sp6-{micro}HC is transported to...DiscussionReferences |
|---|
5 gene. Eur. J. Immunol. 23:1284.[Medline]
. Science 272:411.[Abstract]
5 triple-deficient mice show impaired B cell development but functional allelic exclusion of the IgH locus. J. Immunol. 168:6286. and Ig-. J. Immunol. 169:865.5 protein in B cell development. Cell 69:823.[Medline]
) transgene, but not V()J() gene segment targeted into the Ig() locus, can rescue B cell development in 5-deficient mice. Immunity 5:229.[Medline]
5-deficient mice. J. Immunol. 167:3734.5 surrogate L chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice. Cell 77:133.[Medline]
from the ER/Cis-Golgi. J. Immunol. 171:3091.
5 protein. Eur. J. Immunol. 31:1999.[Medline]
This article has been cited by other articles:
|
|
 |
|
  A. B. Eberle, K. Herrmann, H.-M. Jack, and O. Muhlemann Equal transcription rates of productively and nonproductively rearranged immunoglobulin {micro} heavy chain alleles in a pro-B cell line RNA, June 1, 2009; 15(6): 1021 - 1028. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. B. Nakajima, K. Kiefer, A. Price, G. C. Bosma, and M. J. Bosma Two Distinct Populations of H Chain-Edited B Cells Show Differential Surrogate L Chain Dependence J. Immunol., March 15, 2009; 182(6): 3583 - 3596. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Vettermann, K. Herrmann, C. Albert, E. Roth, M. R. Bosl, and H.-M. Jack A Unique Role for the {lambda}5 Nonimmunoglobulin Tail in Early B Lymphocyte Development J. Immunol., September 1, 2008; 181(5): 3232 - 3242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Achour, P. Cavelier, M. Tichit, C. Bouchier, P. Lafaye, and F. Rougeon Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus J. Immunol., August 1, 2008; 181(3): 2001 - 2009. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Geraldes, M. Rebrovich, K. Herrmann, J. Wong, H.-M. Jack, M. Wabl, and M. Cascalho Ig Heavy Chain Promotes Mature B Cell Survival in the Absence of Light Chain J. Immunol., August 1, 2007; 179(3): 1659 - 1668. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Meister, U. Schubert, K. Neubert, K. Herrmann, R. Burger, M. Gramatzki, S. Hahn, S. Schreiber, S. Wilhelm, M. Herrmann, et al. Extensive Immunoglobulin Production Sensitizes Myeloma Cells for Proteasome Inhibition Cancer Res., February 15, 2007; 67(4): 1783 - 1792. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Fuentes-Panana, G. Bannish, F. G. Karnell, J. F. Treml, and J. G. Monroe Analysis of the Individual Contributions of Ig{alpha} (CD79a)- and Igbeta (CD79b)-Mediated Tonic Signaling for Bone Marrow B Cell Development and Peripheral B Cell Maturation J. Immunol., December 1, 2006; 177(11): 7913 - 7922. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zou, J. A. Smith, V. K. Nguyen, L. Ren, K. Luyten, S. Muyldermans, and M. Bruggemann Expression of a Dromedary Heavy Chain-Only Antibody and B Cell Development in the Mouse J. Immunol., September 15, 2005; 175(6): 3769 - 3779. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. B. Guloglu, E. Bajor, B. P. Smith, and C. A. J. Roman The Unique Region of Surrogate Light Chain Component {lambda}5 Is a Heavy Chain-Specific Regulator of Precursor B Cell Receptor Signaling J. Immunol., July 1, 2005; 175(1): 358 - 366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. J. Vosshenrich, A. Cumano, W. Muller, J. P. Di Santo, and P. Vieira Pre-B cell receptor expression is necessary for thymic stromal lymphopoietin responsiveness in the bone marrow but not in the liver environment PNAS, July 27, 2004; 101(30): 11070 - 11075. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Corcos, H.-M. Jack, W. Schuh, S. Meister, and E. Roth Signaling and Cell Surface Expression of {micro}H Chains in the Absence of Light Chains J. Immunol., January 1, 2004; 172(1): 4 - 5. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, 
) and Rag2- mice (
, 
), all of which had received tetracycline in the drinking water for 7 days, were cultured without (closed symbols) and with tetracycline (open symbols) over a period of 4 days in the presence (+ IL-7) or absence (-IL-7) of 2% IL-7 supernatant. Cell numbers (No.) were determined by flow cytometry. Mean values and SEs were calculated from triplicates. The data represents one of three independently performed experiments.