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The Journal of Immunology, 2001, 167: 1920-1928.
Copyright © 2001 by The American Association of Immunologists

Signaling Through TNF Receptor p55 in TNF-{alpha}-Deficient Mice Alters the CXCL13/CCL19/CCL21 Ratio in the Spleen and Induces Maturation and Migration of Anergic B Cells into the B Cell Follicle1

Laura Mandik-Nayak*,{dagger}, Guangming Huang*, Kathleen C. F. Sheehan*, Jan Erikson{ddagger} and David D. Chaplin2,*,{dagger}

* Center for Immunology and {dagger} Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110; and {ddagger} The Wistar Institute, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The organization of secondary lymphoid tissues into distinct T and B cell compartments supports proper regulation of an immune response to foreign Ags. In the splenic white pulp, this compartmentalization is also thought to be important in the maintenance of B cell tolerance. Using lymphotoxin-{alpha}-(LT-{alpha})-, TNF-{alpha}-, or TNFRp55-deficient mice, all with disrupted splenic architecture, we tested whether normal T/B segregation and/or intact follicular structure are necessary for the maintenance of anti-dsDNA B cell anergy. This study demonstrates that anti-dsDNA B cells remain tolerant in LT-{alpha}-/-, TNF-{alpha}-/-, and TNFRp55-/- mice; however, TNF-{alpha} or a TNF-{alpha}-dependent factor is required for their characteristic positioning to the T/B interface. Providing a TNF-{alpha} signal in TNF-{alpha}-/- mice by systemic administration of an agonist anti-TNFRp55 mAb induces the maturation of the anti-dsDNA B cells and their movement away from the T cell area toward the B cell area. Additionally, the agonist Ab induces changes in the follicular environment, including FDC clustering, up-regulation of the CXC chemokine ligand CXCL13, and down-regulation of the CC chemokine ligands CCL19 and CCL21. Therefore, this study suggests that a balance between B and T cell tropic chemokine signals may be an important mechanism for positioning anergic B cells at the T/B interface of the splenic white pulp.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cells and B cells are normally segregated within the spleen white pulp into discrete areas known as the periarteriolar lymphoid sheath (PALS)3 and B cell follicle, respectively. The B cell follicle is itself compartmentalized, containing a ring of marginal zone B cells around the periphery of each white pulp nodule, and follicular aggregates of B cells located around clusters of follicular dendritic cells (FDCs). This ordered organization of the splenic white pulp is thought to be important for proper regulation of immune responses to both foreign and self Ags (1, 2). Lymphotoxin-{alpha} (LT-{alpha}) and TNF-{alpha} provide signals that support the generation and maintenance of this lymphoid architecture (3, 4, 5, 6, 7, 8). Mice deficient in TNF-{alpha}, LT-{alpha}, or one of their receptors, TNFRp55, exhibit a range of defects in follicular structure in the spleen. TNF-{alpha}-/- and TNFRp55-/- mice manifest loss of normal structure of the B cell zone. There are no organized clusters of FDCs, and the B cells are distributed in a loose ring around each white pulp nodule (4, 9). LT-{alpha}-deficient mice display a more severe phenotype with an almost complete overlap of T and B cell zones and the absence of FDCs altogether (3, 10, 11).

Recently, several chemokines have been identified that direct the positioning of B and T cells within the splenic white pulp: the CXC chemokine ligand CXCL13 (BLC) (12, 13), and the CC chemokine ligands CCL19 (ELC) (14) and CCL21 (SLC) (15). Consistent with their disrupted splenic architecture, LT-{alpha}-/- mice and TNF-{alpha}-/- mice express lower levels of RNA for these chemokines. Spleens from LT-{alpha}-/- mice have greatly reduced levels of CXCL13, CCL19, and CCL21 RNA, whereas TNF-{alpha}-/- and TNFRp55-/- spleens express lower levels of CXCL13 only (16). The importance of T/B segregation, FDCs, and germinal centers (GCs) in the generation of immune responses to foreign Ags has been well documented (17, 18, 19); however, their role in the initiation and maintenance of B cell tolerance to self Ags has not been investigated.

Ig transgene (Tg) models of B cell tolerance have revealed several fates for anti-dsDNA B cells, including deletion (20), receptor editing (21), and anergy (22, 23). Presumably, these distinct manifestations of tolerance reflect differences in Ag recognition. A consistent observation regarding anergic anti-dsDNA B cells, as well as anergic B cells in another tolerance model, is that they are excluded from the B cell follicle (22, 24). Conversely, in lupus-prone MRL-lpr/lpr mice, in which B cell tolerance breaks down, the anti-dsDNA B cells are able to enter the B cell follicle. Furthermore, MRL-lpr/lpr mice lack segregated T and B cell areas and eventually lose conventional B cell follicles as they age and begin producing serum autoantibodies (25). One study has suggested that FDC function may be diminished in older MRL-lpr/lpr mice (26). Interestingly, aged C57BL/6 and BALB/c mice (>15 mo of age) also have mixed T and B cell areas in the spleen and express serum autoantibodies (27).

It is unclear whether desegregation of T cell and B cell areas and a lack of intact follicular structure are integral components of the breakdown of B cell tolerance. To address this, we have bred the VH3H9 IgH chain Tg (a model of anti-DNA B cell tolerance) onto several gene-targeted strains in which normal spleen white pulp microarchitecture is disturbed. In this model, the VH3H9 IgH chain Tg pairs with multiple endogenous IgL chains to generate both anti-DNA and nonanti-DNA Abs (28, 29). VH3H9 in combination with the endogenous Ig{lambda}1 L chain generates an anti-dsDNA specificity (28). This permits tracking of anti-dsDNA B cells in the diverse repertoire of the mouse. Previously, we have shown that VH3H9/{lambda} B cells are actively regulated in wild-type mice, as manifest by their arrest at an immature stage of development, localization to the T/B interface in the spleen, and failure to produce anti-dsDNA Ab in the serum (22). This regulation breaks down in autoimmune MRL-lpr/lpr mice: the VH3H9/{lambda} B cells are no longer developmentally arrested; they localize to the B cell follicle; and their Ab becomes detectable in the serum (25).

In this study, we have crossed the VH3H9 Tg onto LT-{alpha}-/-, TNF-{alpha}-/-, and TNFRp55-/- backgrounds and tested whether tolerance of the transgenic anti-dsDNA B cells remains intact. This study shows that neither normal segregation of T and B cells nor an intact B cell follicular structure is necessary for inducing and/or maintaining B cell anergy to dsDNA, as the VH3H9/{lambda} B cells remain developmentally arrested and do not produce Ab. Interestingly, the characteristic positioning of anergic B cells to the T/B interface in the spleen is dependent on TNF-{alpha} or a TNF-dependent factor. Signaling through TNFRp55 in TNF-{alpha}-/- mice using an agonist Ab induces the maturation of VH3H9/{lambda} B cells and their movement into the B cell follicle. Additionally, this agonist Ab signals FDCs to move from their abnormal position in the MZ to cluster in a more normal distribution in the B cell area. These changes in movement and maturation correlate with an increase in splenic RNA for the B cell tropic chemokine CXCL13 and decrease for the T cell tropic chemokines CCL19 and CCL21. Taken together, these findings suggest a mechanism for the localization of anergic B cells to the T/B interface that is dependent on a balance between CXCL13 and CCL19/CCL21 signals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). LT-{alpha}-/- mice (3) and TNFRp55-/- mice (gift of J. Peschon, Immunex, Seattle, WA) (30) have been backcrossed onto the C57BL/6 background for 10 generations. TNF-{alpha}-/- mice (gift of M. Marino and L. Old, Ludwig Institute for Cancer Research, New York, NY) (31) have been maintained on a C57BL/6 x 129 mixed background. VH3H9 Tg mice (32) have been backcrossed >15 generations onto the BALB/c background and are maintained as hemizygotes with respect to the Tg. The mice have been bred and maintained in a separate specific pathogen-free room at The Division of Comparative Medicine at the Washington University School of Medicine (St. Louis, MO) animal facility according to protocols approved by the institutional committee for the humane use of experimental animals. To obtain VH3H9 LT-{alpha}-/-, TNFRp55-/-, or TNF-{alpha}-/- mice, VH3H9 BALB/c mice were crossed to knockout mice to obtain VH3H9 mice that were heterozygous for the targeted allele. These VH3H9 heterozygotes were then crossed back to homozygous knockout mice to obtain VH3H9 mice that were homozygous for the knockout allele. Mice used were between 6 and 20 wk of age. Heterozygote littermates of the knockout mice and age-matched BALB/c mice, housed similarly, were used as controls. In all cases, heterozygote and wild-type mice were indistinguishable. Therefore, in the figures, only wild-type mice are shown as controls. The presence of the VH3H9 Tg and the homozygous presence of the LT-{alpha}-, TNFRp55-, or TNF-{alpha}-targeted alleles were determined by PCR amplification of tail DNA with primers specific for VH3H9 (32), LT-{alpha} (LT-{alpha}1, LT-{alpha}2, NeoA, and NeoB), TNF-{alpha} (TNF-{alpha}1, TNF-{alpha}2, NeoA, and NeoB), and TNFRp55 (p55B, P55E, p55-spe, and pgk-66), respectively. Primer sequences are as follows: LT-{alpha}1, 5'-CTA GCT AAC TCA GAG TCC TAG AGT-3'; LT-{alpha}2, 5'-TTA CCA ACA AGG TGA GCA GCA GGT-3'; TNF-{alpha}1, 5'-CAG TTC TAT GGC CCA GAC CCT C-3'; TNF-{alpha}2, 5'-CTC AGC CAC TCC AGC TGC TC-3'; NeoA, 5'-ATC GCA TCG AGC GAG CAC GTA CTC GGA-3'; NeoB, 5'-AGC TCT TCA GCA ATA TCA CGG GTA GCC-3'; p55B, 5'-GGA TTG TCA CGG TGC CGT TGA AG-3'; p55E, 5'-TGA CAA GGA CAC GGT GTG TGG C-3'; p55-spe, 5'-TGC TGA TGG GGA TAC ATC CAT C-3'; pgk-66, 5'-CCG GTG GAT GTG GAA TGT GTG-3'.

Determination of lymphocyte phenotype by flow cytometry

Cells (5 x 105) were surface stained according to standard protocols (33). The following Abs were used: RA3-6B2 PE or biotin (anti-B220), R11-153 FITC (anti-Ig{lambda}1), R26-46 FITC or biotin (anti-Ig{lambda} total), 1D3 FITC (anti-CD19), 7G6 FITC (anti-CD21/35), Cy34.1 FITC (anti-CD22), B3B4 PE (anti-CD23), IM7 PE (anti-CD44), and M1/69 FITC (anti-CD24, heat-stable Ag (HSA)) (BD PharMingen, San Diego, CA); JC5.1 PE (anti-Ig{lambda} total) (gift from J. Kearney, University of Alabama, Birmingham, AL); polyclonal anti-IgG FITC (Sigma, St. Louis, MO), polyclonal anti-IgM PE, and SBA-1 PE (anti-IgD) (Southern Biotechnology Associates, Birmingham, AL). Streptavidin-Red670 was obtained from Life Technologies (Gaithersburg, MD).

All samples were analyzed using a FACScan flow cytometer (BD Biosciences, Mountain View, CA) with CellQuest software. Gating on live lymphocytes based on forward and side scatter, 40,000 events were collected for each sample.

Identification of VH3H9/{lambda}1 anti-dsDNA B cells

The VH3H9 Tg encodes an IgM H chain only. It has been shown to be an effective excluder of endogenous H chain rearrangement in the BALB/c background (32). To verify that there was no detectable use of endogenous, nontransgenic H chain, we examined B cells for surface IgD and IgG (which would necessarily be encoded by endogenous H chains), and none were detected (data not shown). VH3H9 when paired with Ig{lambda}1 generates an anti-dsDNA Ig. This permits us to follow the fate of anti-dsDNA B cells in VH3H9 Tg mice using anti-Ig{lambda}-specific reagents (22). Several different reagents were used to track Ig{lambda}+ and Ig{lambda}1+ B cells (R11-153, JC5.1, and R26-46). Using these reagents and flow cytometry, we have shown that the majority (>95%) of Ig{lambda}+ B cells in VH3H9 and VH3H9 LT-{alpha}-/-, TNFRp55-/-, and TNF-{alpha}-/- mice are Ig{lambda}1, as they are in Tg- mice (22 ; data not shown). Therefore, we are able to follow VH3H9/{lambda}1 B cells in LT-{alpha}-/-, TNFRp55-/-, and TNF-{alpha}-/- mice using anti-pan Ig{lambda} reagents.

Immunohistochemistry

Spleens were suspended in OCT, frozen in 2-methylbutane cooled with liquid nitrogen, sectioned at 8 µm, and fixed with acetone. The spleen sections were stored at -20°C and then stained according to the protocol as described (17). Briefly, endogenous peroxidase was quenched using 0.3% hydrogen peroxide, nonspecific binding was blocked using PBS/5% normal goat serum (Sigma)/0.1% Tween 20, and then the sections were stained with RA3-6B2 biotin (anti-B220), GK1.5 biotin or FITC (anti-CD4), 8C12 biotin (anti-CD35), anti-rat Ig biotin (BD PharMingen), FDC-M2 (anti-FDC; gift of M. Kosco-Vilbois, Serono Pharmaceutical Research Institute, Geneva, Switzerland), MOMA-1 (anti-marginal zone macrophage; Serotec, Oxford, U.K.), and/or goat anti-Ig{lambda} alkaline phosphatase (AP; Southern Biotechnology Associates). Complement-coated immune complex binding was detected using mouse peroxidase anti-peroxidase (Vector Laboratories, Burlingame, CA) preincubated with fresh mouse serum at 37°C for 10 min as a source of complement. Mouse peroxidase anti-peroxidase without serum was used as a control to test for the necessity of complement to detect the immune complex binding. Streptavidin-HRP or AP (Southern Biotechnology Associates), or anti-FITC AP (Sigma) was used as the secondary Ab. HRP and AP were developed using the substrates 3-amino-9-ethyl-carbazole and Fast-Blue BB base (Sigma), respectively.

The percentage of VH3H9/{lambda} B cells localized to the T cell area vs B cell area was determined by counting the number of Ig{lambda}+ B cells in the T cell area (B220-CD4+ by immunohistochemistry) and dividing by the total number of Ig{lambda}+ B cells in the white pulp area (B220+ and CD4+). At least two follicles were counted per mouse without knowledge of mouse genotype.

Detection of anti-nuclear Ab (ANA)

The presence of ANAs in serum samples was detected using permeabilized HEP-2 cells as the substrate following the manufacturer’s instructions (Antibodies, Davis, CA). Sera giving a homogeneous nuclear staining pattern were defined as ANA+. This pattern is found in a high frequency of systemic lupus erythematosus serum and correlates with the presence of anti-dsDNA, anti-histone, and/or anti-chromatin Abs (34). Serum samples were used at a 1/100 dilution. ANA binding was detected using a goat anti-mouse IgM + goat anti-mouse IgG or Ig{lambda} FITC secondary Abs (Southern Biotechnology Associates). The samples were visualized under a fluorescent microscope and scored without knowledge of mouse genotype.

Ab injections

Mice were injected i.v. with 50 µg 55R-593 (hamster anti-TNFRp55) or PIP-1D1 (hamster Ig control Ab) (gifts of R. Schreiber, Washington University) on days 0 and 4 and sacrificed on day 7. The optimal dose and frequency of injections were tested in a titration experiment (data not shown). The 55R-593 Ab does not cross-react with the TNFRp75 and has been shown to have agonist activity against the TNFRp55 in vitro (35).

Northern blots

A total of 20 µg total RNA from the spleens of anti-TNFRp55 Ab-treated or control Ab-treated TNF-{alpha}-/- or wild-type mice was separated by gel electrophoresis, transferred to Hybond N+ membranes (Amersham Pharmacia Biotech, Piscataway, NJ), and probed using randomly primed 32P-labeled mouse cDNA probes. The probes were: CXCL13, nt 33–360; CCL21, nt 28–462; and CCL19, nt 177–625. To control for loading, the blots were rehybridized using mouse {beta}-actin cDNA as a probe. To quantitate the bands, Northern blots were developed using a Storm 840 PhosphorImager, and the data were analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Chemokine RNA levels were normalized by dividing the signal for each chemokine sample by the {beta}-actin signal for that sample.

Statistical analysis

Statistical significance was determined using an unpaired nonparametric test and Instat Software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Altered localization, but not altered surface phenotype or Ab secretion, of VH3H9/{lambda} B cells in TNF-{alpha}-/-, TNFRp55-/-, and LT-{alpha}-/- spleens

To determine whether a lack of T/B segregation and/or intact follicular structure would interfere with the establishment or maintenance of B cell tolerance to self Ags, we bred the VH3H9 Ig Tg tolerance model onto LT-{alpha}-/-, TNF-{alpha}-/-, and TNFRp55-/- mice. In wild-type mice, VH3H9/{lambda} B cells are localized at the interface between the T and B cell areas in the spleen as well as in the red pulp (Fig. 1GoA) (22). LT-{alpha}-/- mice have overlapping T and B cell areas, making it difficult to define distinct T and B cell areas; however, where T cell areas can be distinguished, the VH3H9/{lambda} B cells localize with the T cells (Fig. 1GoA). TNF-{alpha}-/- and TNFRp55-/- mice lack clusters of FDCs in the B cell follicle, but do have segregated T and B cell areas (4, 9). This allows us to test the necessity of intact follicular structure in the positioning of anergic B cells. In TNF-{alpha}-/- and TNFRp55-/- mice, the VH3H9/{lambda} B cells are found spread throughout the PALS (percentage of Ig{lambda}+ B cells in PALS: TNF-{alpha}-/-, 89.1 ± 2%; TNFRp55-/-, 94.2 ± 2.6%), whereas the majority of Ig{lambda}+ B cells are at the T/B interface in BALB/c mice (61.7 ± 3.9% in the periphery of the T cell zone). Therefore, TNF-{alpha}- or LT-{alpha}-dependent white pulp structure is required for the segregation of anti-dsDNA B cells to the T/B interface. Ig{lambda} B cells in Tg- wild-type, TNF-{alpha}-/-, TNFRp55-/-, and LT-{alpha}-/- mice are found with the rest of the B cells in the mouse, showing that there is nothing inherently different about the localization of Ig{lambda} B cells. Rather, the effects noted above are due to the autoreactive specificity of VH3H9/{lambda} (Fig. 1GoB).



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  		FIGURE 1. Altered localization of VH3H9/{lambda} B cells in TNF-{alpha}-/-, TNFRp55-/-, and LT-{alpha}-/- mice. Serial spleen sections from VH3H9 Tg (A) and Tg- (B) mice were stained with Abs against B220 (top) or CD4 (bottom) in red and Ig{lambda} in blue. Images are representative sections from six mice of each genotype. Original magnification x100.

 
Because the VH3H9/{lambda} B cells in the knockout mice did not localize within the white pulp in a pattern typical of anergic cells, we considered that either TNF-{alpha}, LT-{alpha}, or the structures they specify might be required for normal tolerance of these cells. One of the features of tolerance defined for VH3H9/{lambda} B cells is developmental arrest (22), as determined by the relative expression levels of several cell surface markers, including B220, HSA, CD21/35, CD22, and CD44 (33, 36, 37, 38, 39, 40, 41). We first demonstrated that B cells from Tg- wild-type, TNF-{alpha}-/-, TNFRp55-/-, and LT-{alpha}-/- mice express mature B cell levels of these markers, and therefore are, by this analysis, phenotypically normal (data not shown). VH3H9/{lambda} B cells are present in spleens at similar frequencies in wild-type (14.6 ± 3.8%), TNF-{alpha}-/- (15.5 ± 5.3%), TNFRp55-/- (17.9 ± 1.2%), and LT-{alpha}-/- (17.8 ± 4.1%) with the same 4- to 5-fold decreased level of surface Ig. Furthermore, VH3H9/{lambda} B cells in TNF-{alpha}-/-, TNFRp55-/-, and LT-{alpha}-/- mice, like those in wild-type mice, are phenotypically immature (B220low, CD21/35low, CD22low, HSAhigh, and CD44high; data not shown). We and others have correlated the down-regulation of surface Ig with Ag encounter (22, 23, 42, 43, 44, 45, 46, 47). Therefore, although deficiency in TNF-{alpha}, TNFRp55, or LT-{alpha} affects the splenic localization of VH3H9/{lambda} B cells, it does not prevent their encounter with self Ag, nor alter their developmental arrest. Furthermore, Ig{lambda} ANAs were not present in the serum from VH3H9 TNF-{alpha}-/-, TNFRp55-/-, or LT-{alpha}-/- mice (Table IGo). Together with the lack of change in surface phenotype, this suggests that despite their abnormal white pulp localization, the tolerance of VH3H9/{lambda} B cells remains intact in mice deficient in TNF-{alpha}, TNFRp55, or LT-{alpha}.


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  		Table I. Serum ANA status1

 
LT-{alpha}-/-, TNF-{alpha}-/-, and TNFRp55-/- mice express serum ANAs

Although the anergic VH3H9/{lambda} anti-dsDNA B cells, with or without LT-{alpha}, TNF-{alpha}, or TNFRp55, do not secrete autoantibodies, serum ANAs are detected in some of the knockout mice (Table IGo). Presumably, these autoantibodies originate from anti-dsDNA B cells that are normally tolerized by a different mechanism other than anergy (20, 21, 48, 49, 50). TNF-{alpha}-/- mice with and without the VH3H9 Tg have detectable serum ANAs, whereas only the TNFRp55-/- and LT-{alpha}-/- mice with the VH3H9 Tg were ANA+ (Table IGo). Hybridoma analysis has demonstrated that the VH3H9 Tg increases the frequency of ANA B cells in the spleen (29). It is possible, then, that the frequency of ANA B cells in Tg- TNFRp55-/- and LT-{alpha}-/- mice is too low to detect and that the increased frequency of autoreactive B cells in VH3H9 Tg mice is what allows for detection of their Ab in the serum.

Signaling through TNFRp55 repositions VH3H9/{lambda} B cells in TNF-{alpha}-/- mice

VH3H9/{lambda} B cells are aberrantly located in the PALS of TNF-{alpha}-/- and TNFRp55-/- mice. To determine what factors dictate this altered localization, we tested whether inducing a signal through TNFRp55 in vivo would lead to repositioning of the autoreactive B cells. VH3H9 and Tg- TNF-{alpha}-/- mice, along with VH3H9 and Tg- wild-type mice, were injected i.v. with an agonist anti-TNFRp55 Ab or a control Ab. Seven days later, spleens were harvested and assayed for the localization of the VH3H9/{lambda} B cells (Fig. 2Go). In control Ab-injected VH3H9 TNF-{alpha}-/- mice, the Ig{lambda}+ B cells are localized in the PALS (90.8 ± 4.6% in PALS) as they are in unmanipulated VH3H9 TNF-{alpha}-/- mice (compare Fig. 2GoA with Fig. 1GoA). In striking contrast, in anti-TNFRp55-treated TNF-{alpha}-/- mice, the VH3H9/{lambda} B cells move away from the center of the PALS and are now found both at the T/B interface and within the B cell area (10.3 ± 3.9% in PALS; Fig. 2GoA). Within the B cell area, VH3H9/{lambda} B cells are also found in the marginal zone, as demarcated by MOMA-1 staining (data not shown). The anti-TNFRp55 Ab has no effect on the localization of Ig{lambda}+ B cells in Tg- TNF-{alpha}-/- or wild-type mice (Fig. 2Go, B and D). Additionally, the localization of VH3H9/{lambda} B cells in anti-TNFRp55-treated wild-type mice is indistinguishable from control Ab-injected mice (Fig. 2GoC). This demonstrates that treatment with the anti-TNFRp55 Ab replaces a signal affecting anergic B cells that is missing in TNF-{alpha}-/- mice.



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  		FIGURE 2. Agonist anti-TNFRp55 Ab alters the splenic localization of VH3H9/{lambda} B cells in TNF-{alpha}-/- mice. VH3H9 TNF-{alpha}-/- (A), Tg- TNF-{alpha}-/- (B), VH3H9 wild-type (C), and Tg- wild-type (D) mice were injected i.v. with 50 µg anti-TNFRp55 Ab or hamster Ig control Ab on days 0 and 4. Spleens were harvested on day 7, and serial sections were stained with Abs against B220 (top) or CD4 (bottom) in red and Ig{lambda} in blue. At this magnification, some of the Ig{lambda}+ cells appear to stain with a greater intensity. When examined under a higher magnification, the Ig{lambda}+ cells are surface Ig{lambda}+ and not cytoplasmic Ig{lambda}+, indicating that they are not plasma cells, consistent with the serum-negative status of the mice. Representative sections from five mice of each genotype per condition are shown. Original magnification x100.

 
VH3H9/{lambda} B cells overcome their developmental arrest in agonist anti-TNFRp55 Ab-treated TNF-{alpha}-/- mice

Treatment of TNF-{alpha}-/- mice with agonist anti-TNFRp55 Ab induces VH3H9/{lambda} B cells to relocate from the PALS toward the B cell area (Fig. 2GoA). To determine whether this relocation also alters their developmental status, the surface phenotype of VH3H9/{lambda} B cells from anti-TNFRp55 Ab-treated mice was compared with that from control Ab-treated mice (Fig. 3Go). VH3H9/{lambda} B cells from control Ab-injected mice are developmentally arrested similar to those in untreated TNF-{alpha}-/- and wild-type mice (Fig. 3Go) (22). In contrast, the VH3H9/{lambda} B cells from anti-TNFRp55 Ab-treated TNF-{alpha}-/- mice are phenotypically mature. They are B220high, HSAlow, CD22high, and CD44low (Fig. 3Go). CD21/35 (Fig. 3Go) and surface Ig (data not shown) levels remain decreased. Decreased CD21/35 and surface Ig levels in the context of increases in other markers of maturity were seen previously in VH3H9/{lambda} B cells from autoimmune VH3H9 MRL-lpr/lpr mice and have been attributed to these cells’ continued encounter with Ag (25). The surface phenotype of Ig{lambda} B cells from Tg- TNF-{alpha}-/- mice treated with anti-TNFRp55 Ab was indistinguishable from those in mice treated with control Ab (Fig. 3Go). During this 7-day Ab treatment, no VH3H9/{lambda} anti-dsDNA Ab was detected in the serum of control or agonist Ab-injected TNF-{alpha}-/- mice (data not shown). Although these short-term experiments cannot address whether prolonged treatment of TNF-{alpha}-/- mice with the agonist anti-TNFRp55 Ab would abrogate tolerance of the VH3H9/{lambda} B cells, they do provide an experimental model to investigate signals that control the localization of the anergic B cells within the white pulp.



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  		FIGURE 3. Agonist anti-TNFRp55 Ab alters the developmental arrest of VH3H9/{lambda} B cells in TNF-{alpha}-/- mice. VH3H9 and Tg- TNF-{alpha}-/- mice were injected i.v. with 50 µg anti-TNFRp55 Ab or hamster Ig control Ab on days 0 and 4. Spleens were harvested on day 7, and cells were isolated and stained with Abs to B220, Ig{lambda}, and either HSA, CD21/35, CD22, or CD44. Histograms are gated on B220+Ig{lambda}+ B cells from anti-TNFRp55 Ab-treated mice (bold lines) overlaid those from hamster Ig control Ab-treated mice (thin lines). B cells from an untreated wild-type mouse are shown as a control for the mature B cell level of staining (top panel). Plots are representative of five mice of each genotype per condition. The percentage of VH3H9/{lambda} B cells did not significantly change in agonist vs control Ab-treated TNF-{alpha}-/- mice (agonist Ab, 10.1 ± 2%; control Ab, 13.6 ± 2.2%, p > 0.1). VH3H9/{lambda} B cells from agonist Ab-treated TNF-{alpha}-/- mice appear phenotypically mature, in that they express increased levels of B220 (mean fluorescence intensity change, {Delta}MFI 167.7 ± 70.9) and CD22 ({Delta}MFI 14.6 ± 4.7), and decreased levels of HSA ({Delta}MFI 297.2 ± 21.9) and CD44 ({Delta}MFI 70.3 ± 5.6). Levels of CD21/35 remain unaltered ({Delta}MFI 0.53 ± 3.1).

 
Systemic administration of agonist anti-TNFRp55 Ab induces clustering of FDCs in TNF-{alpha}-/- mice

To begin to decipher what factors might be affecting the relocalization of VH3H9/{lambda} B cells in TNF-{alpha}-/- mice treated with the agonist anti-TNFRp55 Ab, we investigated what other features of the splenic architecture changed with this Ab treatment. Previous studies have shown that FDCs are aberrantly localized to the marginal zone in TNF-{alpha}-/- mice (Fig. 4Go) (51). FDCs are necessary for GC development, which supports high affinity somatically mutated B cell responses (19). Because these responses are diminished or missing in TNF-{alpha}-/- mice (4, 31), it has been speculated that either the altered location of FDCs or a failure of their maturation leads to a defect in their function (51). To determine whether treatment of TNF-{alpha}-/- mice with the agonist anti-TNFRp55 Ab influences the location of FDCs, spleens from agonist Ab-treated and control Ab-treated TNF-{alpha}-/- mice were analyzed by immunohistochemistry. FDCs were marked with Abs against CD35. FDCs in wild-type mice are clustered within the B cell follicle. In contrast, the CD35 staining in control Ab-treated TNF-{alpha}-/- mice is localized in a ring surrounding the B cell area. Strikingly, in agonist anti-TNFRp55 Ab-treated TNF-{alpha}-/- mice, the CD35 staining is clustered in the B cell area (Fig. 4Go). Because CD35 is also expressed on B cells, albeit at much lower levels than on FDCs, we confirmed that the CD35+ cells detected in the TNF-{alpha}-/- mice were indeed FDCs by FDC-M2 staining (data not shown) and by their ability to bind immune complexes (Fig. 4Go). Furthermore, typical of the immune complex binding to FDCs via CD21/35, this binding was dependent on the presence of complement. Control sections incubated with immune complexes formed in the absence of complement showed no detectable staining (Fig. 4Go).



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  		FIGURE 4. Agonist anti-TNFRp55 Ab induces the relocalization of FDCs. TNF-{alpha}-/- mice were injected i.v. with 50 µg anti-TNFRp55 Ab or hamster Ig control Ab on days 0 and 4. Spleens were harvested on day 7, and serial sections were stained with Abs against CD4 (blue) and either CD35 (top), peroxidase/anti-peroxidase + serum for immune complex deposition (middle), or peroxidase/anti-peroxidase without serum to control for complement dependence of immune complex deposition (bottom) (red). These images are representative of five mice for each condition. Original magnification x100.

 
Systemic anti-TNFRp55 Ab induces increased CXCL13 and decreased CCL19 and CCL21 levels in TNF-{alpha}-/- mice

Treatment of TNF-{alpha}-/- mice with the agonist anti-TNFRp55 Ab leads to the movement of FDCs from the periphery of the B cell zone into clusters and also induces the relocation of VH3H9/{lambda} B cells from the PALS to the B cell follicle. Together, these data suggest that the chemokine environment within the B cell area has changed in agonist Ab-treated mice. Untreated TNF-{alpha}-/- mice have decreased spleen expression of RNA encoding the B cell tropic chemokine CXCL13 (Fig. 5Go) (16). To determine whether treatment with the agonist Ab up-regulated CXCL13, we measured CXCL13 RNA levels from TNF-{alpha}-/- mice treated with either agonist anti-TNFRp55 Ab or control Ab and compared that with levels found in similarly treated wild-type mice (Fig. 5Go). As has been previously reported, TNF-{alpha}-/- spleens express decreased levels (3- to 4-fold lower) of CXCL13 RNA compared with wild-type spleens. Agonist anti-TNFRp55 Ab treatment caused the up-regulation of CXCL13 RNA levels 1.5-fold. CXCL13 levels were also increased 1.5- to 2-fold in wild-type mice treated with the agonist Ab. Although the level of CXCL13 RNA in agonist anti-TNFRp55 Ab-treated TNF-{alpha}-/- mice did not reach the levels found in control Ab-treated wild-type mice (only 50% of the wild-type levels), the increase in CXCL13 RNA in agonist Ab-treated TNF-{alpha}-/- mice does correlate with the movement of both VH3H9/{lambda} B cells to the B cell zone and FDCs into clusters in the B cell follicle. It is intriguing that the increased CXCL13 levels do not cause a similar migration of VH3H9/{lambda} B cells in wild-type mice. One explanation may be that the chemokine threshold for CXCL13 has already been reached.



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  		FIGURE 5. Agonist anti-TNFRp55 Ab induces the up-regulation of CXCL13 and down-regulation of CCL19 and CCL21 RNA in TNF-{alpha}-/- mice. A, Representative Northern blots of total splenic RNA from anti-TNFRp55 Ab-treated or control Ab-treated mice probed to detect CXCL13, CCL19, or CCL21. {beta}-actin was used as a control for RNA loading. B, Relative chemokine RNA levels as determined by phosphorimager analysis of the Northern blots after correction for RNA loading using the corresponding hybridization with a {beta}-actin probe. Values on the graph depict the mean percentage ± SEM as calculated by dividing the values for individual agonist Ab-treated mice after correction for RNA loading by the mean value for control-treated mice after correction for RNA loading. The values for control Ab-treated mice are set to 100% (dotted line). n = 4 mice of each genotype for each condition.

 
In addition to an increased level of CXCL13, which could act to attract the VH3H9/{lambda} B cells toward the B cell follicle, we also considered that treatment with the agonist anti-TNFRp55 Ab might induce a concomitant decrease in the T cell tropic chemokines CCL19 and CCL21. In this scenario, the VH3H9/{lambda} B cells enter the B cell follicle due to an absence of a signal to stay in the T cell area (i.e., by default). To address this, CCL19 and CCL21 RNA levels were compared in agonist and control Ab-treated mice. Both CCL19 and CCL21 levels are decreased 1.5- to 2-fold in anti-TNFRp55 Ab compared with control Ab-treated TNF-{alpha}-/- mice. CCL21, but not CCL19, levels are decreased 1.5-fold in wild-type animals treated with agonist Ab (Fig. 5Go). Therefore, the relocation of VH3H9/{lambda} B cells in anti-TNFRp55 Ab-treated TNF-{alpha}-/- mice may be due to multiple factors, including an increase in expression of CXCL13 and decrease in expression of CCL19 and CCL21. Together, these data suggest that a balance between B cell area tropic and T cell area tropic chemokines is responsible for the localization of anergic anti-dsDNA B cells to the T/B interface in wild-type spleens.


   Discussion
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 Abstract
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 Materials and Methods
 Results
 Discussion
References
 
In this study, we have used VH3H9 Tg mice deficient in LT-{alpha}, TNF-{alpha}, or TNFRp55 to investigate the role that organized splenic white pulp plays in the generation and maintenance of anti-dsDNA B cell anergy. In the VH3H9 Tg model, anergic VH3H9/{lambda} B cells have been characterized both functionally and phenotypically. Functionally, they are unable to secrete Ab in vivo and are refractory to stimulation in vitro (52). Phenotypically, they are developmentally arrested, have a rapid in vivo turnover rate, and fail to enter the B cell follicle in the spleen (22). Previously, we have used a bcl-2 Tg to separate the rapid turnover rate from these other features of anergy (49). Until now, it has been unclear whether exclusion from the B cell follicle is required for the maintenance of B cell tolerance in the context of a diverse B cell repertoire. To address this, we have bred the VH3H9 Tg onto mice that maintain T/B segregation, but lack intact B cell follicles (TNF-{alpha}-/- and TNFRp55-/-), and onto mice with severely overlapping B and T cell areas (LT-{alpha}-/-). This study demonstrates that VH3H9/{lambda} anti-dsDNA B cells remain tolerant in LT-{alpha}-/-, TNF-{alpha}-/-, and TNFRp55-/- mice, in that they do not secrete autoantibodies and they maintain an immature phenotype. It is striking that in TNF-{alpha}-/- and TNFRp55-/- mice, in which T and B cell areas are segregated, the anergic B cells are scattered throughout the PALS rather than being located at the T/B interface. Therefore, neither localization to the T/B interface, nor the overall segregation of T and B cells into discrete areas is necessary for the initiation and/or maintenance of anergic B cell tolerance.

Studies using different B cell tolerance models have demonstrated that anergic B cells localize to the T/B interface in the spleen (22, 24, 53). A working model to account for the localization of these anergic cells is that it is the consequence of an aborted immune response. After encountering Ag, B cells normally migrate to the interface between the T and B cell areas (18, 54, 55). Here, presumably, they receive T cell help and then either further differentiate into Ab-secreting cells or migrate back to the follicle to induce GC formation (17). When T cell help is absent due to tolerance in the T cell compartment, the B cells do not differentiate, but remain at the T/B interface with a shortened lifespan (52, 56). The signals that direct Ag-engaged B cells to the T/B interface are most likely gradients of the chemokines CXCL13, CCL19, and CCL21. In support of this, activated, but not naive B cells have increased in vitro responsiveness to the T cell area tropic chemokines CCL19 and CCL21 (14, 57, 58). In TNF-{alpha}-/- mice, CXCL13 levels are reduced, disturbing the chemokine gradient (16). As a result, we postulate that the anergic B cells do not stop at the T/B interface, but rather spread into and throughout the PALS.

Replacement of the TNFRp55 signal using an agonist Ab induced several changes in TNF-{alpha}-/- mice. The first was that the follicular environment changed. In untreated TNF-{alpha}-/- mice, FDCs are aberrantly located in a ring at the marginal zone (51). In mice treated with the agonist Ab, the FDCs cluster in the B cell area and resemble those found in wild-type mice. This structural change in the B cell follicle is accompanied by changes in the expression of chemokines that are known to direct the migration of B and T cells in the spleen. TNF-{alpha}-/- mice express reduced levels of CXCL13, but near normal levels of CCL21 and CCL19 (16). This chemokine balance is altered in anti-TNFRp55 Ab-treated mice. CXCL13 levels are up-regulated, and CCL19 and CCL21 levels are down-regulated. These results are in contrast to studies using TNFRp55-Ig fusion proteins to block signaling through TNFRp55 in vivo that did not show appreciable changes in lymphoid architecture or lymphocyte migration in adult mice (6, 59). However, chemokine levels were not tested. These studies using the blocking fusion protein suggested that the TNFRp55 signal is required during development to set up the splenic environment, and that once set up, the TNF signal is not needed to maintain the architecture. Our data, using the agonist anti-TNFRp55 Ab, are not necessarily inconsistent with this hypothesis. It is possible that the spleen in TNF-{alpha}-/- mice, unlike the spleen in wild-type mice, is not developmentally mature. The agonist Ab, then, would induce the maturation of the spleen. In fact, we see evidence for the development of more normal splenic architecture after anti-TNFRp55 Ab treatment in terms of FDC clustering and increased CXCL13 levels. Alternatively, these studies using the agonist Ab may define a previously unrecognized plasticity of splenic structure (at least in TNF-{alpha}-/- mice).

In addition to inducing changes in white pulp structure, treatment of TNF-{alpha}-/- mice with agonist anti-TNFRp55 Ab also led to maturation of the anergic anti-dsDNA B cells and their migration into the B cell follicle. This demonstrates that the factor(s) involved in holding the anti-dsDNA B cells in the PALS at an immature stage of development in TNF-{alpha}-/- mice is overridden by signaling through the TNFRp55. It is unclear which TNFRp55-expressing cell(s) is receiving the agonist Ab signal. Most cells in the spleen, including T cells, B cells, and dendritic cells, express TNFRp55 (60). Thus, the agonist Ab could be acting directly on the B cell, signaling it to mature and relocate. Alternatively, the agonist anti-TNFRp55 Ab could signal a stromal cell, which then alters the follicular environment, making it more attractive for the anergic B cells and FDCs. The agonist Ab had no effect on the migration or maturation of anti-dsDNA B cells in wild-type mice. This, together with the alterations in CXCL13, CCL19, and CCL21 RNA levels in anti-TNFRp55 Ab-treated mice, leads us to favor the hypothesis that the agonist Ab signals primarily to alter the white pulp environment.

In summary, this study demonstrates that B cell tolerance does not depend on the segregation of T and B cells in the spleen; however, TNF-{alpha}-dependent factors are required for the establishment of the follicular environment that maintains the localization of anergic B cells to the T/B interface. Induction of a TNFRp55-mediated signal with the agonist Ab leads to the maturation of the anergic B cells and their migration into the B cell follicle. Furthermore, treatment with the agonist Ab leads to changes in the follicular environment, including clustering of FDCs, increased CXCL13 levels, and decreased CCL19 and CCL21 levels. Together, these data suggest a mechanism for the segregation of anergic B cells to the T/B interface that involves a balance between the chemokines CXCL13, CCL19, and CCL21.


   Acknowledgments
 
We thank Dr. Mitchell Grayson for critical review of the manuscript, Carlene Zindl for making the Northern blot probes, Dr. Sudhir Nayak for help with the graphics, and Eric Ford for genotyping the mice and technical assistance. We also acknowledge the generous contributions from Dr. Robert Schreiber for the anti-TNFRp55 and hamster Ig control Abs, Dr. Marie Kosco-Vilbois for the FDC-M2 Ab, Dr. Jacques Peschon for the TNFRp55-/- mice, and Dr. Michael Marino and Dr. Lloyd Old for the TNF-{alpha}-/- mice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI34580-05 (to D.D.C.). D.D.C. is an investigator of the Howard Hughes Medical Institute. Back

2 Address correspondence and reprint requests to Dr. David D. Chaplin, Department of Microbiology, University of Alabama, 845 19th Street South, Birmingham, AL 35294. E-mail address: david_chaplin{at}microbio.uab.edu Back

3 Abbreviations used in this paper: PALS, periarteriolar lymphoid sheath; ANA, anti-nuclear Ab; AP, alkaline phosphatase; FDC, follicular dendritic cell; GC, germinal center; HSA, heat-stable Ag; LT-{alpha}, lymphotoxin-{alpha}; Tg, transgene; CCL, CC chemokine ligand; CXCL, CXC chemokine ligand. Back

Received for publication April 9, 2001. Accepted for publication June 5, 2001.


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