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From the Department of Dermatology,* Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; the Department of Dermatology, Nagasaki University Graduate School of Biomedical Science, Nagasaki, Japan; and the Department of Immunology, Duke University Medical Center, Durham, North Carolina
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Abstract |
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43%) the development of skin fibrosis, autoantibody production, and hypergammaglobulinemia. B-cell depletion also restored a more normal balance between Th1 and Th2 cytokine mRNA expression in the skin. By contrast, B-cell depletion did not affect skin fibrosis, hypergammaglobulinemia, and autoantibody levels in adult mice with established disease. Thereby, B-cell depletion during disease onset suppressed skin fibrosis, indicating that B cells contribute to the initiation of systemic sclerosis pathogenesis in tight-skin mice but are not required for disease maintenance.
The tight-skin (Tsk/+) mouse serves as a model for SSc,14
with increased synthesis and accumulation of collagen and other extracellular matrix proteins in the skin.15
Homozygous mice die in utero, whereas heterozygous Tsk/+ mice survive but develop skin fibrosis.16
A tandem duplication within the fibrillin-1 gene causes tissue hyperplasia and the SSc-like phenotype.17-21
However, there is also an immunological component to disease pathogenesis because CD4+ T cells, mast cells, and other immunocompetent cells contribute to skin fibrosis in Tsk/+ mice.22-25
For example, the infusion of bone marrow and spleen cells from tight-skin mice into normal mice induces a Tsk-like cutaneous phenotype and autoantibodies.22,26
Importantly, the adoptive transfer of enriched B or T cells alone into normal syngeneic mice does not cause skin fibrosis, whereas B cells alone increased autoantibody production.22
T helper 2 (Th2) cells and T-cell-secreted profibrogenic cytokines contribute specifically to the fibrotic processes in scleroderma.23,27,28
By contrast, CpG oligodeoxynucleotide or interleukin (IL)-12 inhibit skin sclerosis in Tsk/+ mice by stimulating a Th1 immune response.29,30
Transforming growth factor (TGF)-ß and IL-4 may contribute directly to skin fibrosis because they induce hyperresponsive collagen production in Tsk/+ fibroblasts.28,31
Reciprocally, skin fibrosis is prevented in Tsk/+ mice bearing IL-4, IL-4 receptor, STAT6, or TGF-ß gene mutations or by the administration of anti-IL-4 monoclonal antibody (mAb) to newborn Tsk/+ mice.27,28,32-34
Disrupting IL-4 rescues mice homozygous for the tight-skin mutation from embryonic death and also diminishes TGF-ß production by fibroblasts.33
The phenotypic characteristics of SSc patients and Tsk/+ mice are similar, except Tsk/+ mice have pulmonary emphysema and cardiac hypertrophy14
that are not inhibited by anti-IL-4 mAb, the absence of CD4+ T cells, or IL-4, IL-4 receptor, TGF-ß, or Stat6 deficiencies.23,27,32,34
Tsk/+ B cells also display a hyperresponsive phenotype, with enhanced CD19-induced [Ca2+]i responses, higher levels of CD19 tyrosine phosphorylation,13,35
impaired CD22 regulation of signal transduction,36
and the production of autoantibodies against SSc-specific target autoantigens, such as topoisomerase I, RNA polymerase I, and fibrillin-1.37,38
There is also a correlation between the concentration of serum anti-topoisomerase I autoantibodies in Tsk/+ mice and histological and biochemical alterations in the skin.39
Likewise, human autoantibodies to fibrillin-1 activate normal human fibroblasts in culture through the TGF pathway to recapitulate the scleroderma phenotype.10
Thereby, CD19 deficiency in Tsk/+ mice down-regulates B-cell function, improves skin sclerosis (36%), and inhibits autoimmunity.35
However, whether B cells initiate, contribute to disease progression, or play a role in the maintenance of established disease remains unresolved.
B-cell depletion using CD20 mAb-based immunotherapy is an effective treatment for rheumatoid arthritis and other autoimmune diseases,40 but it has not been examined in SSc patients in which current therapies do not improve or suppress the development of skin sclerosis. Likewise, therapeutic strategies that improve skin sclerosis in adult Tsk/+ mice with disease have not been identified. Therefore, a role for B cells in SSc pathogenesis was assessed in Tsk/+ mice using mouse anti-mouse CD20 mAbs that provide a preclinical test for B-cell depletion immunotherapy that is amenable to mechanistic studies and genetic manipulation.41-44
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Materials and Methods |
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Tsk/+ and wild-type littermates were generated by crossing Tsk/+ males (C57BL/6; Jackson Laboratory, Bar Harbor, ME) with wild-type females. CD20–/– mice were as described.41 Female mice were given 5 to 250 µg of sterile IgG2a (MB20-11) anti-mouse CD20 mAb41 or isotype-matched control mAb in PBS. In most cases, mice were injected with mAb every 2 weeks, for a total of four treatments. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Committee on Animal Experimentation of Kanazawa University Graduate School of Medical Science or the Duke University Animal Care and Use Committee.
Flow Cytometry Analysis
Antibodies used included phycoerythrin (PE)-conjugated CD19 (MB19-1)45 mAb; fluorescein isothiocyanate-conjugated B220, CD3, CD4, CD8, and NK1.1 mAbs; and Cy5-conjugated mAb (all from BD Pharmingen, San Diego, CA). Thy1.2 mAb (Caltag, Burlingame, CA) was used for T-cell enumeration. For mouse CD20 expression, biotin-conjugated mouse anti-mouse CD20 (MB20-18)41 mAb was used, with biotin-conjugated anti-human CD20 (B1) mAb (Beckman-Coulter, Hialeah, FL) used as a negative control and streptavidin PE used as a developing reagent (Southern Biotechnology, Birmingham, AL). For two- or three-color immunofluorescence analysis, single-cell lymphocyte suspensions (0.5 to 1 x 106) were stained at 4°C using predetermined optimal concentrations of mAb for 20 minutes as described.46 Cells with the forward and side light scatter properties of lymphocytes were analyzed on a FACScan flow cytometer (BD Immunocytometry Systems, San Diego, CA).
Enzyme-Linked Immunosorbent Assays (ELISAs)
Anti-nuclear antibodies were assessed using HEp-2 substrate cells (Medical and Biological Laboratories, Nagoya, Japan) as described.11
Antibody and autoantibody levels were determined by ELISA as described.11,47
Topoisomerase I antibody levels were quantified using Medical and Biological Laboratories ELISA kits. Serum interferon (IFN)-
and IL-4 levels were quantified using R&D Systems ELISA kits (Minneapolis, MN). Recombinant peptide representing the proline-rich C-terminal region (amino acids 395 to 446) of fibrillin-1 was generated as described9,38
and used as the substrate for fibrillin-1-specific ELISAs as described.35
Skin Fibrosis and Tissue Pathology
Skin was taken from the para-midline, lower back region as full thickness sections extending down to the body wall musculature. Tissues were fixed in 10% formaldehyde solution for 24 hours, embedded in paraffin, cut into sections, stained with hematoxylin and eosin (H&E), and independently examined by three investigators in a blinded manner. Hypodermal thickness, defined by depth of the subcutaneous loose connective tissue layer (ie, the hypodermis or superficial fascia) beneath the panniculus carnosus, was measured for multiple transverse perpendicular sections using an ocular micrometer. Dermal thickness was defined as the thickness of skin from the top of the granular layer to the junction between the dermis and subcutaneous fat. Ten random measurements were taken per tissue section. To quantify hydroxyproline (collagen) content, 6-mm punch biopsies from shaved dorsal skin were treated with chloroform/methanol (2:1, v/v) to remove fat, dried by centrifugation under vacuum, weighed, acid-hydrolyzed for 24 hours at 110°C, dried, redissolved in 200 µl of water, and filtered through Millipore filters. Samples (20-µl aliquots) were diluted 10-fold and analyzed for composition in an amino acid analyzer (Hewlett-Packard, Palo Alto, CA). Lungs inflated after harvesting and hearts were fixed in 10% formaldehyde solution, embedded in paraffin, and stained with H&E.
Cytokine Expression
B-cell-enriched splenocyte preparations (
93% B220+) were generated by depleting Thy1.2 mAb-coated cells using magnetic beads (Dynal Biotech, Oslo, Norway). Total RNA from full-thickness skin sections, splenocyte suspensions, or B16 F10 mouse melanoma cells (American Type Culture Collection, Manassas, VA) was converted into cDNA using a reverse transcription system (Promega, Madison, WI). Transcript levels were quantified by real-time polymerase chain reaction (PCR) using sequence-specific primers and probes designed by pre-developed TaqMan assay reagents (one cycle of 50°C for 2 minutes, 95°C for 10 minutes; 40 cycles of 92°C for 15 seconds, 60°C for 60 seconds) and an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, CA). The relative expression of target transcripts and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript PCR products was determined using the 
Ct method48
using cytokine mRNA levels in wild-type mice as calibrators in triplicate assays with the mean Ct used for calculations.
Statistical Analysis
The Mann-Whitney U-test was used for verifying significant differences between sample means and Bonferroni’s test was used for multiple comparisons.
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Results |
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Mature CD20+ B cells are significantly reduced in wild-type mice by day 2 after intravenous treatment with CD20 mAb (MB20-11, 250 µg/mouse).42,43
We therefore determined whether subcutaneous, intravenous, and intraperitoneal treatment depleted B cells to the same extent throughout a range of mAb concentrations in 2-month-old wild-type mice. Single mAb doses as low as 5 µg given intravenously, intraperitoneally, or subcutaneously depleted most mature bone marrow, blood, spleen, and lymph node B cells after 7 days as determined by immunofluorescence staining with flow cytometry analysis (Figure 1A
; data not shown). Small numbers of CD19low B220+ B cells persisted within the blood, spleen, and lymph nodes after CD20 mAb treatment (Figure 1B)
. Such cells predominantly represented cells with a pre-B/immature B-cell phenotype and were likely to be recent emigrants from the bone marrow as described.43
However, subcutaneous, intravenous, or intraperitoneal CD20 mAb treatments did not significantly alter the frequency or apparent composition of these residual B220+ B-cell populations. After CD20 mAb (5 µg) treatment, the pharmacokinetics of CD20 mAb treatments varied slightly during the first 15 to 60 minutes after mAb administration depending on the route of administration, but mAb clearance and B-cell reconstitution were similar between subcutaneous and intravenous treatments, with increased numbers of circulating and tissue B220+ cells first observed at 2 weeks (not shown; Figure 1C
).
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2 months of age.14,49
When 3-day-old wild-type and Tsk/+ mice were treated with MB20-11 mAb (subcutaneously, 10 µg/mouse), mature CD19+B220+ spleen B-cell numbers were reduced by 85 to 90% 7 days later. Treating 8-week-old wild-type and Tsk/+ littermates with MB20-11 mAb (20 µg s.c.) reduced CD19+ B220+ B-cell numbers by 94 to 98% in the bone marrow, blood, spleen, and lymph nodes 7 days later, while circulating and tissue T-cell numbers were unchanged. Isotype-control mAb treatments did not affect circulating or tissue B-cell numbers. Therefore, B-cell depletion was effective in Tsk/+ littermates when CD20 mAb was given subcutaneously at low doses.
The effectiveness of long-term B-cell depletion in newborn Tsk/+ mice was assessed by MB20-11 mAb (10 or 20 µg) treatment on day 3 after birth, with repeated treatment on days 17, 31, and 45. At 8 weeks of age, the majority of circulating, spleen, and lymph node CD19+ B220+ B cells were depleted to similar extents in both Tsk/+ and wild-type littermates (85 to 99%; Figure 1B
and Table 1
), without affecting T-cell numbers or the relative ratios of circulating or spleen CD4+/CD8+ T cells (data not shown). Small numbers of B220+ B cells persisted in Tsk/+ and wild-type littermates even with 20-µg mAb doses, 1 to 3% for blood and 5 to 6% for spleen and lymph nodes (Table 1)
. The residual B cells present in both Tsk/+ and wild-type littermates predominantly represented B220+ cells with a CD19low pre-B/immature B-cell phenotype (Figure 1B)
as described.43
Thus, B-cell depletion was effective for the entire treatment period, with no indication of differences in CD20 mAb effectiveness between Tsk/+ and wild-type littermates. Furthermore, B-cell depletion had no obvious effects on the normal development, maturation, or connective tissues of juvenile mice because no morphological changes or alterations in movement or development were detected in mice as determined by observation and microscopic examination of tissue sections.
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Although mouse CD20 expression by B-cell subsets has been examined extensively, its expression by other leukocyte subsets has only been partially assessed.41
Moreover, small subpopulations of human T cells and NK cells have been reported to express CD20 at low levels as determined by immunofluorescence staining with flow cytometry analysis.50-53
Therefore, CD20 expression by mouse T cells and NK cells was assessed. B220+ cells represented the predominant, if not exclusive, source of CD20 expression among blood leukocytes from wild-type mice, whereas there was no mAb reactivity with leukocytes from CD20-deficient mice (Figure 1D)
. CD4+ T cells, CD8+ T cells, and NK1.1+ cells did not express CD20 at detectable levels in wild-type mice. In fact, CD20 mAb staining was equivalent for T and NK cells from wild-type and CD20-deficient mice. Thus, CD20 expression appears to be B-cell restricted, consistent with the selective depletion of B cells after mAb treatment.
B-Cell Depletion Suppresses Skin Fibrosis in Tsk/+ Mice
The benefit of B-cell depletion in newborn Tsk/+ mice was assessed by MB20-11 or control mAb (10 or 20 µg) treatment on day 3 after birth, with repeated treatments on days 17, 31, and 45. Skin fibrosis in B-cell-depleted Tsk/+ mice was then assessed by histopathology of skin sections from 56-day-old mice. In Tsk/+ mice, hypodermal thickness was increased eightfold because of hyperplasia of the subcutaneous loose connective tissue layer (Figures 2 and 3A)
as reported.35
Dermal thickness from the top of the granular layer to the junction between the dermis and subcutaneous fat was similar between control mAb-treated Tsk/+ (50 ± 4 µm) and wild-type (52 ± 5 µm) littermates. Subcutaneous fat and panniculus carnosus thickness was also similar between Tsk/+ and wild-type littermates. However, CD20 mAb treatment reduced mean dermal and hypodermal thickness in Tsk/+ mice by 31% (10 µg, P < 0.05) and 43% (20 µg, P < 0.01), respectively, compared with control mAb treatment, although these tissues were significantly thicker than those of control mAb-treated wild-type littermates (P < 0.01, Figure 3A
). B-cell depletion in wild-type mice did not affect dermal or hypodermal thickness.
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). However, CD20 mAb treatment (20 µg) reduced hydroxyproline content by half in Tsk/+ mice (P < 0.01), although skin hydroxyproline content remained significantly higher than in wild-type littermates (P < 0.05). Hydroxyproline content was also significantly (P < 0.05) reduced in Tsk/+ mice treated with 10 µg of CD20 mAb. Despite this, CD20 mAb treatment (10 or 20 µg) did not significantly affect the onset or severity of pulmonary emphysema in Tsk/+ mice because their alveoli remained three to four times larger than in control mice, as described.32
Thus, early B-cell depletion suppressed skin fibrosis in Tsk/+ mice. B-Cell Depletion Does Not Reverse Skin Fibrosis in Tsk/+ Mice
Whether untreated or treated with control mAb, Tsk/+ mice developed significant skin fibrosis and maximal skin thickness by 56 days of age (Figures 2 and 3A
; data not shown). To identify time points in disease progression when B-cell depletion could inhibit or reverse disease, 14-, 28-, and 56-day-old Tsk/+ mice were treated with CD20 mAb (20 µg) bi-weekly until days 70, 84, or 112. Thickness and hydroxyproline content of skin from Tsk/+ mice was significantly reduced when they were treated with CD20 mAb from day 14 until day 70 (Figure 3B
, P < 0.05). However, there was no significant difference in skin thickness for mice treated during days 28 to 84 (Figure 3C)
. Thus, B-cell depletion remained effective for reducing skin fibrosis between days 14 and 28. To assess whether B-cell depletion could reverse disease, 56-day-old Tsk/+ mice were treated with CD20 mAb on days 56, 70, 84, and 98. By day 112, B-cell depletion was equally effective in wild-type and Tsk/+ littermates, with small numbers of CD19low B220+ B cells persisting in Tsk/+ and wild-type littermates, 2% for blood, 3 to 4% for spleen, and 5 to 6% for lymph nodes (Table 1)
. Because the extent of B-cell depletion was similar, if not identical, between Tsk/+ and wild-type littermates (Table 1)
there was no indication of differences in CD20 mAb effectiveness between mice with and without disease. CD20 mAb treatment did not affect T-cell numbers (data not shown). Regardless, CD20 mAb treatment did not significantly affect fibrosis or the hydroxyproline content of skin biopsies from 112-day-old Tsk/+ mice (Figure 3D)
. Likewise, B-cell depletion did not significantly affect emphysema severity in Tsk/+ mice (not shown). Myocardial hypertrophy in 4-month-old Tsk/+ and wild-type mice was indistinguishable from that of mice with comparable heart weights (129 ± 15 mg versus 118 ± 14 mg, respectively; n = 5). CD20 mAb treatment did not affect heart size or total weight in wild-type or Tsk/+ mice (119 ± 12 mg versus 125 ± 13 mg, respectively; n = 5). Thus, B-cell depletion can suppress the development of skin sclerosis early after disease development but does not alter established sclerosis or emphysema in Tsk/+ mice.
B-Cell Depletion Inhibits Autoantibody Generation in Tsk/+ Mice
Although a pathogenic role for autoantibodies in SSc development remains uncertain,10
autoantibody and serum immunoglobulin levels were assessed in Tsk/+ mice as markers for effective B-cell depletion and hyperactivity. B-cell depletion starting on day 3 significantly inhibited both IgM and IgG anti-fibrillin 1 autoantibody production in 56-day-old mice treated with 20 µg of CD20 mAb (Figure 4A)
. However, IgM or IgG anti-fibrillin 1 autoantibody levels were not significantly altered when B-cell depletion was initiated in 56-day-old mice (Figure 4B)
. Serum anti-nuclear antibody generation was also assessed in 56-day-old Tsk/+ and wild-type littermates treated repeatedly with CD20 mAb since day 3 after birth. Anti-nuclear antibodies with a homogenous chromosomal staining pattern were detected in 33% (5 of 15) of Tsk/+ mice treated with control mAb. However, anti-nuclear antibodies were rarely detected in B-cell-depleted Tsk/+ mice (20 µg of CD20 mAb, 1 of 15; 10 µg, 1 of 15), or wild-type mice treated with CD20 (20 µg, 0 of 15) or control (20 µg, 1 of 15) mAb. Similarly, 56-day-old Tsk/+ mice treated with control mAb produced significantly higher levels of rheumatoid factor and topoisomerase I- or single-stranded DNA-specific autoantibodies when compared with control mAb-treated wild-type littermates (P < 0.05, Figure 4C
). However, rheumatoid factor and autoantibody levels were decreased significantly in B-cell-depleted Tsk/+ mice (10 or 20 µg of CD20 mAb) relative to control mAb-treated Tsk/+ littermates when assessed by ELISA (Figure 4C)
or immunoprecipitation assays (data not shown). By contrast, initiating B-cell depletion in 56-day-old Tsk/+ mice did not significantly affect rheumatoid factor or anti-topoisomerase I and anti-DNA autoantibody levels by day 112 (Figure 4D)
. Thus, B-cell depletion dramatically inhibited autoantibody production in Tsk/+ mice when started early, but not after disease development.
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. Tsk/+ mice treated with control mAb had significantly elevated IgM, IgG1, IgG2a, and IgG2b levels compared with wild-type littermates (P < 0.05). However, Tsk/+ mice treated with CD20 mAb (10 or 20 µg) had significantly decreased IgM, IgG1, IgG2a, IgG2b, and IgG3 levels compared with control mAb-treated Tsk/+ littermates. In fact, IgG2a (P < 0.05), IgG2b (P < 0.01), and IgG3 (P < 0.001) levels in CD20 mAb-treated Tsk/+ mice were lower than those in control mAb-treated wild-type littermates. By contrast, B-cell depletion did not affect serum antibody levels in Tsk/+ or wild-type mice when CD20 mAb treatment was started 56 days after birth (Figure 4F)
. Thus, B-cell depletion inhibited the development of hypergammaglobulinemia in young Tsk/+ mice but did not reverse established hypergammaglobulinemia. Skin B-Cell Depletion in Tsk/+ Mice
B-cell-associated transcripts are up-regulated in lesional skin from SSc patients.6
It was therefore assessed whether CD20 mAb-mediated skin B-cell depletion might influence skin fibrosis in young Tsk/+ mice. Tsk/+ and wild-type littermates were treated with CD20 or control mAb (20 µg) at 3 and 17 days, with B-cell-associated transcripts quantified in skin from 3-week-old mice, a time in which blood and spleen CD19+ B220+ B cells were depleted by > 93%. Transcripts for CD19, CD20, and light chains was compared with CD138 (syndecan-1) and T-cell CD3 transcript levels. CD138 is expressed by plasma cells, epithelial cells, vascular smooth muscle cells, endothelium, and neural cells. However, B- and T-cell transcripts were not detectable in the skin of Tsk/+ and wild-type littermates by real-time PCR analysis compared with water-only control reactions or RNA from melanoma cells (Table 2
, not shown). By contrast, CD138 transcripts were readily detected in the skin, while spleens and B-cell-enriched splenocytes expressed high levels of CD19, CD20, 
, 
, and CD3 transcripts. Thus, B cells or plasma cells were not found in the skin of young Tsk/+ mice, even in the absence of CD20 mAb treatment.
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Whether B-cell depletion within lymphoid tissues influenced proinflammatory cytokines critical for the development of skin sclerosis in Tsk/+ mice27,30,34
was assessed by real-time PCR analysis. Tsk/+ and wild-type littermates were treated with either CD20 or control mAb (20 µg) at 3 and 17 days with mRNA expression quantified in skin from 3-week-old mice. In the skin, TGF-ß mRNA levels were elevated in Tsk/+ mice (183 ± 15%) relative to wild-type littermates (100 ± 10, P < 0.01, n = 5; Figure 5A
). However, B-cell depletion significantly reduced TGF-ß transcript levels in Tsk/+ mice (P < 0.05) to 134 ± 13% of wild-type littermate levels, but did not reduce TGF-ß levels in wild-type mice (92 ± 9%). IL-4, IL-6, and IL-10 mRNA levels were increased by 70, 84, and 56%, respectively, in control mAb-treated Tsk/+ mice compared with wild-type littermates (P < 0.01, Figure 5A
). By contrast, TNF-
(44%), IL-2 (31%), and IFN- (29%) mRNA levels were significantly decreased in Tsk/+ mice compared with wild-type littermates (P < 0.01). However, B-cell depletion significantly reduced IL-4, IL-6, and IL-10 mRNA levels in Tsk/+ (P < 0.01) and wild-type (P < 0.01) littermates. B-cell depletion also significantly increased TNF-, IL-2, and IFN- mRNA levels in Tsk/+ (P < 0.05), but not wild-type littermates. Thus, B-cell depletion significantly altered skin cytokine profiles in young Tsk/+ mice.
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mRNA levels were decreased by 48, 66, and 45%, respectively, in control mAb-treated Tsk/+ mice compared with wild-type littermates (P < 0.01, Figure 5B
). By contrast, IL-6 (76%), IL-10 (68%), and TNF- (29%) mRNA levels were significantly increased in Tsk/+ mice (P < 0.05), compared with wild-type littermates. CD20 mAb-induced B-cell depletion significantly increased IL-4 (84%), IL-2 (59%), and IFN- (79%) mRNA levels in Tsk/+ mice (P < 0.05) but also resulted in increased IL-4, IL-6, IL-10, TNF-, IL-2, and IFN- mRNA levels in wild-type (P < 0.05) littermates. By contrast, TGF-ß mRNA expression was not significantly elevated in Tsk mice (126 ± 12%) relative to wild-type littermates (100 ± 15, n = 5). Furthermore, B-cell depletion did not significantly affect TGF-ß transcript levels in either wild-type or Tsk/+ littermates. Although increased cytokine transcript levels in Tsk/+ and wild-type littermates may result from the increased ratio of T cells to B cells, these results document that B-cell depletion nonetheless significantly altered the cytokine profiles in spleens of young Tsk/+ mice. To determine whether B-cell depletion altered the systemic balance of Th1/Th2 cytokines, serum levels of IFN- (representative Th1 cytokine) and IL-4 (representative Th2 cytokine) were measured. However, neither cytokine was detectable (n = 10 mice for each group), consistent with earlier findings of others that it is not possible to detect IL-4 in Tsk/+ mouse serum.34
Thus, B-cell depletion differentially affected skin and spleen cytokine levels, which may have therapeutic benefits. |
Discussion |
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. B-cell depletion also rebalanced skin cytokine transcript levels in Tsk/+ littermates with progressing sclerosis and significantly altered spleen cytokine transcript levels (Figure 5)
. Although the extent of B-cell depletion was similar in newborn and adult Tsk/+ mice (Table 1)
, sclerosis, autoantibody levels, and hypergammaglobulinemia in mice with established disease was not altered by CD20 mAb-mediated B-cell depletion (Figures 2 and 4)
. Thus, B cells significantly influenced cutaneous fibrosis during disease development but were not necessary for the maintenance of disease in Tsk/+ mice.
Demonstrating that B-cell depletion reduces skin fibrosis in Tsk/+ mice provides a basis for identifying the mechanisms through which B cells may influence autoimmune disease. B cells can produce autoantibodies, influence cytokine expression and promote Th2 cell development, function as antigen-presenting cells, and contribute to local inflammation. Although theoretical and not observed in the current studies, B-cell depletion in young wild-type and Tsk/+ mice might also affect skin development, which is not yet complete at 3 days after birth. Early B-cell depletion in Tsk/+ mice also prevented autoantibody production and hypergammaglobulinemia, whereas B-cell depletion in adult Tsk/+ mice with disease did not reduce autoantibody levels or hypergammaglobulinemia (Figure 4)
. Preventing the generation of autoreactive B cells and the production of anti-fibrillin-1 antibodies plus other autoantibodies may be important because correlations exist between the concentration of serum anti-topoisomerase I autoantibodies and histological and biochemical alterations in the skin of Tsk/+ mice.39
Autoantibodies to fibrillin-1 also activate cultured human fibroblasts to recapitulate the fibrotic SSc phenotype in vitro.10
Although autoantibodies correlate with B-cell hyperactivity and disease in Tsk/+ mice, further studies are needed to determine whether autoantibodies contribute directly to disease in vivo.
The influence of B-cell depletion on skin and spleen cytokine levels provides the most likely mechanistic explanation for B-cell depletion affecting skin fibrosis. B-cell depletion reduced skin TGF-ß transcript levels and also reduced IL-4, IL-6, and IL-10 mRNA expression in young Tsk/+ mice, while significantly increasing TNF-, IL-2, and IFN- production (Figure 5A)
. This may explain reduced skin fibrosis because Th2 cytokines such as IL-4 and IL-6 stimulate the synthesis of extracellular matrix proteins in dermal fibroblasts,54,55
whereas Th1 cytokines such as IFN- and TNF- suppress fibroblast collagen production in vitro.56
A relative shift to Th2 rather than Th1 cytokines can also induce tissue fibrosis.56
Likewise, blocking IL-4 function inhibits skin fibrosis in newborn Tsk/+ mice,27,34
whereas IL-12 production can prevent sclerosis and autoimmunity in Tsk/+ mice.30
Normalized cytokine expression after CD20 mAb treatment did not appear to result from the depletion of skin B cells, because it was difficult to detect an increase in either B or T cells in the skin of Tsk/+ mice by immunohistochemistry (not shown) or by real-time PCR amplification of B- and T-cell transcripts (Table 2)
. Thus, it is likely that B-cell depletion inhibited skin fibrosis by altering cytokine production by T cells circulating through the skin or indirectly affecting serum cytokines. For example, IL-10 produced by activated B cells inhibits IL-12 production by dendritic cells, which promotes Th2 T-cell differentiation.57
In support of this, CD20 mAb treatment significantly altered cytokine transcript levels within the spleens of treated mice (Figure 5B)
, and may thereby alter the central and peripheral balance of Th1/Th2 cells. Consistent with this, numerous studies have documented a role for IL-4 produced postnatally in skin hyperplasia and indicated that CD4+ T cells of the Th2 phenotype influence the skin manifestations of the scleroderma-like syndrome in Tsk/–mice.28
Moreover, normalizing tissue IL-4 levels may reduce tissue fibrosis induced by TGF-ß, because IL-4 induces TGF-ß gene expression and IL-4 and TGF-ß interact in regulating collagen gene expression in vitro.32,33
At least two mechanisms for B-cell depletion affecting T-cell cytokine production are possible. First, B-cell depletion may affect T cell-B cell interactions that would normally promote Th2 cell generation and/or cytokine production. Alternatively, the initial depletion of most peripheral B cells followed by the continued removal of maturing B cells as they emerge from the bone marrow may induce activation of monocytes/macrophages within CD20 mAb-treated mice and thereby alter their maturation, regulatory functions, and/or cytokine production. Thus, B-cell depletion in Tsk/+ mice may have pleiotrophic effects on the lymphocyte networks that influence T cell function and cytokine production within lymphoid tissues and influence skin-infiltrating inflammatory cells.
The current results are consistent with studies showing that B cells do not cause skin fibrosis in Tsk/+ mice18,49,58
but do contribute to disease pathogenesis.22,26
Although some studies have concluded that B cells play no role in skin sclerosis, this interpretation could have resulted from several factors, including small sample sizes,18,58
mixed genetic backgrounds of mice,18,49,58
and reliance on stretchable skin length58
or dermal thickness18
as disease parameters. Dermal thickness was similar in Tsk/+ and wild-type littermates in the current (Figure 2)
and previous studies,14,23,24,49
while hypodermal thickness, including a subcutaneous connective tissue layer, is increased eightfold in Tsk/+ C57BL/6 mice (Figure 2A)
, as reported.24
Hypodermal thickness in Tsk/+ BALB/c mice is only two- or threefold increased, potentially complicating some studies.49
Baxter and colleagues59
have recently confirmed that dermal thickness and dermal collagen content of Tsk/+ and wild-type mouse skin are comparable. Based on this, Baxter and colleagues59
concluded that Tsk/+ mice are not useful for studying anti-fibrotic therapeutics, despite their greater skin-fold thickness. However, the current study and a number of previous studies have already demonstrated normal dermal thickness in Tsk/+ mice, but increased hypodermal thickness,14,23,24,39,49
increased collagen production,25,32,39,60
and augmented TGF-ß mRNA expression22
in the skin. That the sites of fibrosis are different between human SSc (dermis) and Tsk/+ mice (hypodermis) may represent species-specific differences in skin architecture. Thus, although results from Tsk/+ mice cannot be simply translated into human therapies, the skin sclerosis and autoimmunity of Tsk mice make this a very useful animal model for skin fibrosis and scleroderma. Because the current model system is not complicated by genetic background differences and does not require adoptive lymphocyte transfers, this strategy suggests the utility of B-cell-targeted therapies in SSc patients with early disease and provides a pathway for mechanistic studies of SSc pathogenesis.
This study indicates that B-cell-directed therapies may benefit SSc patients when given during early stages of disease because skin fibrosis was diminished in Tsk/+ mice after B-cell depletion between days 3 and 14, and before 28 days after birth (Figure 3, A and B)
. Likewise, anti-IL-4 mAb treatments reduce Tsk/+ mouse skin thickening when initiated during the first week after birth.34
Halofuginone, an inhibitor of type-1 collagen synthesis and skin sclerosis, is effective in newborn and 4-week-old Tsk/+ mice.61
Similarly, the development of pulmonary emphysema in Tsk/+ mice was not affected by B-cell depletion or by other immunomodulatory treatments.23,27,32,34
Thus, B cells and other components of the immune system contribute to early disease initiation or progression while established skin sclerosis and pulmonary emphysema are likely to involve additional factors. Because T cells, mast cells, and other immune cells also contribute to skin fibrosis in Tsk/–mice, it remains possible that inhibiting T-cell and mast cell function in addition to B-cell depletion may have additive effects. Small subpopulations of human blood and bone marrow CD4+ and CD8+ T cells, and NK cells have been reported to express CD20 at low levels as determined by immunofluorescence staining with flow cytometry analysis.50-53
Although human T and NK cells have never been shown to produce CD20 transcripts or the CD20 protein directly, these findings have lead to the suggestion that depletion of these cells contributes to the therapeutic benefit of CD20 mAb treatment in humans.53
In mice, circulating CD4+ and CD8+ T cells, or NK cells did not express detectable CD20 cell surface protein (Figure 1D)
. Similarly, mouse bone marrow, blood, spleen, or lymph node leukocytes do not express detectable CD20 as determined by immunofluorescence staining of T cells, NK cells, dendritic cells, or monocytes/macrophages (K. Yanaba, J.-D. Bouaziz, and T.F. Tedder, manuscript in preparation). These results in mice are in agreement with the vast majority of studies assessing CD20 expression by human leukocytes, where CD20 expression has been found to be B-cell-restricted.62
Thus, it remains likely that B-cell depletion may have secondary effects on T cells that influence skin sclerosis.
Although the positive results of this study may not translate to currently available CD20 mAb therapies in humans, the finding that intravenous, subcutaneous, and intraperitoneal CD20 mAb treatments depleted tissue B cells equally in mice (Figure 1)
suggest that low CD20 mAb doses may effectively treat some autoimmune diseases. The 10-µg doses of CD20 mAb (1.5 mg/m2) given to 2-month-old mice (Figure 1)
were 250-fold lower than the 375-mg/m2 doses of rituximab normally given to oncology patients four times during the course of a month. Moreover, SSc patients may benefit from repeated low-dose subcutaneous CD20 mAb treatments given during early disease onset compared with intermittent high-dose mAb infusions. Consistent with this, B-cell depletion was beneficial after early disease onset in Tsk mice (Figure 3B)
. A failure of CD20 mAb treatment to reduce disease or autoantibody levels in adult Tsk/+ mice also mimics findings in numerous human autoimmune diseases after CD20 mAb treatment,40
presumably because of the presence of long-lived CD20-negative plasma cells or incomplete B-cell depletion. It is likely that early B-cell depletion in Tsk/+ mice prevented hypergammaglobulinemia and diminished autoantibody production by eliminating mature B cells before plasma cell differentiation. Alternatively, autoreactive B cells generated later during the course of disease progression may persist after CD20 mAb treatment. Nonetheless, although autoantibodies correlate with B-cell hyperactivity and disease in Tsk/+ mice, further studies are needed to determine whether autoantibodies contribute to disease and to identify the role for B cells in directing T-cell function in vivo. Because no therapy has been proven effective in suppressing or improving skin sclerosis in controlled studies of SSc patients or in Tsk/+ mice with disease manifestations, and CD20 mAb treatment has few side effects,40
therapies that effectively eliminate B cells or simply reduce B-cell hyperactivity during early disease onset may treat SSc or contribute to the therapeutic benefit of treatments yet to be identified.
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Acknowledgements |
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Footnotes |
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Supported by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan; the Uehara Memorial Foundation; Kanazawa University, Japan (to M.H.); the Japan Rheumatism Foundation; Nagasaki University, Japan (to S.S.); the Arthritis Foundation, and the National Institutes of Health (CA105001, CA96547, and AI56363 to T.F.T.).
S.S. and T.F.T. share senior authorship.
Accepted for publication May 16, 2006.
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2 gene on development of cutaneous hyperplasia in tight skin mice. Mol Med 1998, 4:356-360[Medline] and transforming growth factor-ß genes. J Invest Dermatol 2001, 116:136-143[CrossRef][Medline] receptors regulates B lymphocyte depletion during CD20 immunotherapy. J Exp Med 2006, 203:743-753This article has been cited by other articles:
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