Thymic Alterations in GM2 Gangliosidoses Model Mice

Background Sandhoff disease is a lysosomal storage disorder characterized by the absence of β-hexosaminidase and storage of GM2 ganglioside and related glycolipids. We have previously found that the progressive neurologic disease induced in Hexb −/− mice, an animal model for Sandhoff disease, is associated with the production of pathogenic anti-glycolipid autoantibodies. Methodology/Principal Findings In our current study, we report on the alterations in the thymus during the development of mild to severe progressive neurologic disease. The thymus from Hexb −/− mice of greater than 15 weeks of age showed a marked decrease in the percentage of immature CD4+/CD8+ T cells and a significantly increased number of CD4+/CD8− T cells. During involution, the levels of both apoptotic thymic cells and IgG deposits to T cells were found to have increased, whilst swollen macrophages were prominently observed, particularly in the cortex. We employed cDNA microarray analysis to monitor gene expression during the involution process and found that genes associated with the immune responses were upregulated, particularly those expressed in macrophages. CXCL13 was one of these upregulated genes and is expressed specifically in the thymus. B1 cells were also found to have increased in the thy mus. It is significant that these alterations in the thymus were reduced in FcRγ additionally disrupted Hexb −/− mice. Conclusions/Significance These results suggest that the FcRγ chain may render the usually poorly immunogenic thymus into an organ prone to autoimmune responses, including the chemotaxis of B1 cells toward CXCL13.


Introduction
Lysosomal storage disorders (LSDs) arise from functional defects in one or more of the proteins essential to normal lysosome function.This typically involves the enzymes that play a critical role in the intracellular digestion of glycoproteins, glycolipids, glycosaminoglycans, or other macromolecules [1].GM2 gangliosidoses, one of the major LSDs, are caused by an abnormality in the b-hexosaminidases [1,2].b-Hexosaminidase A consists of a heterodimer of an asubunit (HEXA gene product) and a b-subunit (HEXB gene product) whereas b-Hexosaminidase B is a homodimer of b-subunits.Mutations in the HEXA gene cause Tay-Sachs disease, whereas mutations in the HEXB gene cause Sandhoff disease (SD) [1].
Previous studies have shown that the Hexb-deficient (Hexb 2/2 ) mouse develops an SD-like illness and therefore provides a useful animal model for investigating the pathophysiology of SD [3][4][5].As with many of the other LSDs, neurodegeneration is a prominent feature of SD and since the neurons accumulate a large amount of GM2 ganglioside and GA2 relative to other tissues in this disease, it is generally thought that the nervous system is its main pathological target.The accumulation of GM2 ganglioside or its derivatives in the nervous system is implicated in unscheduled neuronal cell death [6].However, recent studies have provided good evidence that GM2 ganglioside and GA2 accumulation can not account for all of the nervous system damage in Hexb 2/2 mice.
We have reported in a previous study that an autoimmune response occurs in Hexb 2/2 mice with accompanying pathophysiological phenotypes [10].To determine the role of antiganglioside autoantibodies in this earlier study, we additionally disrupted the Fc receptor gamma (FcRc) gene in the Hexb 2/2 mouse model, as it plays a key role in immune complex-mediated autoimmune diseases.Clinical symptoms were improved and life spans were extended in the resulting double-null (Hexb 2/2 FcRc 2/2 ) mice, suggesting that autoantibodies play an important role in the central nervous system (CNS) pathophysiology in SD.Moreover, we found that age-matched Hexb 2/2 FcRc 2/2 mice had a reduced serum titer of anti-GA2 autoantibody when compared with Hexb 2/2 FcRc +/+ mice, suggesting that the FcRc dependent pathway(s) also contribute to the production of autoantibodies [10].Recently also, autoantibodies have been implicated in several LSDs and their respective mouse models such as MPSIIIB [11], Batten disease [12][13][14][15], and Gaucher disease [16,17].This suggests that the production of autoantibodies is mediated by common pathway(s) among these diseases although the underlying mechanism of production remains unknown.
The thymus is a central or primary lymphoid organ responsible for the production, differentiation and direction of a population of small lymphocytes that are involved primarily with cell-mediated immunity.Such thymus-dependent lymphocytes are known as Tlymphocytes in contrast to the bone marrow dependent Blymphocytes with which they intimately co-operate.Recently, alterations in the thymus were observed in several LSD model animals such as feline GM1 gangliosidosis [18][19][20], and twitcher mice [21].In Hexb 2/2 mice, GM2 and GA2 were found to have accumulated in the thymus [22].Impaired selection of invariant natural killer T cells was also observed in this same study [22].However, whether such alterations in the thymus contribute to the LSD pathophysiology remains unknown.
Over the last decade, a family of chemotactic cytokines known as the chemokines has been found to be involved in the trafficking of leukocytes in both normal and pathological states.Several autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and myasthenia gravis, are associated with inappropriate activation of the chemokine network [23].For example, aberrant high expression of Chemokine (C-X-C motif) ligand 13 (CXCL13) in the thymus in aged (NZB 6NZW) F1 (BWF1) mice may play a pivotal role in breaking immune tolerance in the thymus and in recruiting autoantibody-producing B1 cells and CD4 + T cells during the development of murine lupus [24][25][26].B1 cells of different origin and function than conventional B cells (B2 cells) have long been considered to be involved in autoantibody production in autoimmune diseases [27][28][29].
In Hexb 2/2 mice, cDNA microarray analysis of the CNS has previously revealed an upregulation of inflammatory cytokines/ chemokines including CXCL13, dominated by activated microglia/macrophages [8].The expected mechanism underlying this microglia/macrophage activation was the storage of glycolipids from engulfed apoptotic neurons.Normally, most T cells are eliminated within the cortex or at the cortico-medullary junction via programmed cell death.These T cells are usually rapidly engulfed by macrophages without accompanying immune activation.It is thus possible that if the macrophages cannot degrade apoptotic T cell-derived glycolipids, they are also activated.
We hypothesize that the thymus plays an important role in the production of autoantibodies in SD.We have therefore examined the thymic alterations of Hexb 2/2 mice during the development of mild to severe progressive neurologic disease in our current study and explored the relationship between these thymic abnormalities and autoimmunity.
Histological examination of the thymus from Hexb +/2 FcRc +/+ , Hexb 2/2 FcRc +/+ and Hexb 2/2 FcRc 2/2 mice We next undertook histological analysis of the thymic tissues from Hexb +/2 FcRc +/+ , Hexb 2/2 FcRc +/+ and Hexb 2/2 FcRc 2/2 mice.When examined by light microscopy, the histological findings from younger Hexb 2/2 FcRc +/+ mice were found not to significantly differ from Hexb +/2 FcRc +/+ mice.In the 15 week old Hexb 2/2 FcRc +/+ mice, the cellular density did not appear to be altered in the medulla of the thymus but the cortices or cortico-medullary junctions were frequently indistinct (Fig. 2).Moreover, many foamy macrophages could be observed not only in the medullary area, but also the cortex area in these mice.Some macrophages were also found to contain hematoxylin-positive nuclear particles in their cytoplasm.Cortices or cortico-medullary junctions were clearly detectable in the thymus of Hexb 2/2 FcRc 2/2 mice and foamy macrophages were also present in Hexb 2/2 FcRc 2/2 mice.We also examined the immunofluorescent analysis of GA2 and thymocyte, for each thymus derived from Hexb +/2 FcRc +/+ , Hexb 2/2 FcRc +/+ and Hexb 2/2 FcRc 2/2 mice.In the 15 week old Hexb +/2 FcRc +/+ mice, GA2 was stained on the cell membrane of CD4 and/or CD8 positive T cells (Fig. S1).Age matched Hexb 2/2 FcRc +/+ mice showed the marked decrease of the T cells in the cortex area.In contrast, GA2 was stained in those areas, and it seems to be accumulating in the cytosol of the foamy cells.The decrease of the T cell and the accumulation of GA2 were not more clearly observed in the presymptomatic stage of Hexb 2/2 FcRc +/+ , and Hexb 2/2 FcRc 2/2 mice.These results suggest the possibility that the localization of GA2 was moved from T cells to foamy macrophages during the involution.

Histological examination of a thymus from a human SD patient
To determine whether thymic alterations can also be detected in a human case of SD, we obtained a H&E stained tissue sample from an SD patient for examination.The histology of this thymic sample revealed that the cortical cells were severely reduced and that the cortico-medullary junctions could not be clearly observed (Fig. 3).Furthermore, most of the cortical and medullary cells harbored a vacuolated cytoplasm.

Macrophage activation and expansion in thymus
The swelling of macrophages is a prominent feature of many LSDs.To confirm whether the swollen cells we observed by H&E staining of our thymic sections were macrophages, we stained these samples with Iba-1 and found that positive cells were rarely observed in the cortices of the 15 week old Hexb +/2 FcR +/+ mice.13 week old Hexb 2/2 FcR +/+ mice were found not to significantly differ from Hexb +/2 FcRc +/+ mice.On the other hand, numerous Iba-1 positive swelling cells were detected in the 15 week old Hexb 2/2 FcR +/+ thymus, particularly in the cortex.In Hexb 2/2 FcR 2/2 mice, at 15 weeks of age, the numbers of both foamy cells and amoeboid, Iba-1-positive thymic macrophages were substantially reduced compared with the Hexb 2/2 FcR +/+ mice (Fig. 5A).To determine whether the percentage of macrophages among the total thymic cell population was increased, we performed flow cytometric analysis.The percentage of thymic macrophages from 15 week old Hexb +/2 FcR +/+ , Hexb 2/2 FcR +/+ and Hexb 2/2 FcR 2/2 mice was 0.9360.38,4.3660.66and 1.4860.25%respectively (Fig. 5B).Recent studies have reported that the up-regulation of the Mip-1a correlates with monocyte infiltration and the pathogenesis of SD [30].To quantify the increase in Mip-1a mRNA in the thymus of 15 week old Hexb 2/2 FcRc +/+ mice, the gene expression ratios of Mip-1a relative to ribosomal protein s18 were measured by real-time RT-PCR.A 24.6-fold increase in Mip-1a expression was detected in the thymus of Hexb 2/2 FcRc +/+ mice in comparison with Hexb +/2 FcRc +/+ mice.In addition, Hexb 2/2 FcRc 2/2 mice showed a 2.69-fold higher expression of Mip-1a mRNA compared with the Hexb +/2 FcRc +/+ mice (Fig. 5C).
mRNA expression profile in the thymus of Hexb 2/2 FcRc +/+ mouse To further elucidate the molecular basis of thymic involution, we performed cDNA microarray analysis to identify the changes in gene expression that accompanied the involution of the thymus.Thymic gene expression of 15 week old Hexb +/2 FcRc +/+ and Hexb 2/2 FcRc +/+ mice were examined with AffymetrixH Mouse Genome 430 2.0 Array containing 45101unique mouse gene sequences.The gene expression profile reported in this paper has been deposited in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo:accession no.GSE19641).8018 probes were found to be relatively increased in the thymus of the Hexb 2/2 FcRc +/+ mice compared with that of the Hexb +/2 FcRc +/+ mice.On the other hand, the expression of 7604 probes was relatively decreased.The cohort of up-regulated sequences was dominated by genes that play a role in the immune response.In addition, some of these genes are expressed in macrophage lineages such as macrophage expressed gene 1, and colony stimulating factor 2 receptor beta 1. Th2 cytokines were mostly upregulated in the Hexb 2/2 FcRc +/+ mice, although Th1 cytokines did not show this increase (Table 1).In addition, B cell-related genes such as CD19, CXCL13 were increased, whereas the T cellrelated genes were mostly decreased, in Hexb 2/2 FcRc +/+ mice compared with Hexb +/2 FcRc +/+ mice.

Discussion
Sandhoff disease is a lysosomal storage disorder characterized by the absence of b-hexosaminidase and storage of GM2 ganglioside and related glycolipids.It is widely postulated that the CNS is the main pathological target of GM2 gangliosidosis.However, we have previously found that the production of autoantibodies plays an important role in the pathogenesis of neuropathy in Sandhoff disease [10].In our present study, we observed a dramatic involution of the thymus in 15 week old Hexb 2/2 FcRc +/+ mouse.The thymus plays a crucial role in immune system homeostasis, and thymic abnormalities have been previously reported in many autoimmune diseases, such as myasthenia gravis [31], systemic sclerosis [32], in a mouse model of systemic lupus erythematosus [24], and multiple sclerosis [33].Since autoimmunity is found also in SD, the contribution of thymus abnormalities to the pathogenesis of this disease seems plausible.We therefore tested for immunological alterations in the thymus of Hexb 2/2 FcR +/+ mice during the development of mild to severe progressive neurologic disease and explored the relationship between thymic abnormalities and autoimmunity.
The thymus of the Hexb 2/2 FcR +/+ mouse was found to be drastically involuted at the late stages, whereas this degree of involution was reduced in the Hexb 2/2 FcRc 2/2 mouse (Fig. 1A,  B), suggesting that this process is dependent on the FcR(chain.The involution itself was found to contain a markedly decreased and Hexb +/2 FcR +/+ mice were stained with antibodies against the macrophage marker Iba1 and processed using the avidin-biotin-peroxidase complex (ABC) method.The bottom panels show higher magnifications of the framed areas in the top panels.Scale bars, top panel, 100(mm; bottom panel, 50 mm.(B) Thymic cells from15 week old Hexb +/2 FcRc +/+ , Hexb 2/2 FcRc +/+ and Hexb 2/2 FcRc 2/2 mice were stained with anti-Iba1 followed by Alexa Fluor 488H conjugated anti-rabbit IgG (H+L), and the percentages of macrophages were determined by flow cytometry (n = 4).*p,0.01.(C) Mip-1a mRNA levels in the thymus from 15week old Hexb 2/2 FcRc +/+ and Hexb 2/2 FcRc 2/2 mice were assayed by Real-time RT-PCR and these values were compared with those of the Hexb +/2 FcRc +/+ mouse (mean 6 S.D.; n = 5-9).*P,0.05.doi:10.1371/journal.pone.0012105.g005percentage of immature CD4+/CD8+ T cells and significantly increased populations of CD4+/CD8-cells, CD4-/CD8+ T cells and CD4-/CD8-cells (Fig. 1C, top panel).The absolute number of each subpopulation was decreased, suggesting that the changes in cell number are strongly dependent on the decreased number of CD4+/CD8+ T cells, rather than the increase in CD4+/CD8cells, CD4-/CD8+ T cells and CD4-/CD8-cells.In contrast, no differences could be clearly observed in the younger Hexb 2/2 FcR +/+ mice.Zhou and co-workers have also shown in their previous study that the development of classical, naive, and memory CD4 and CD8 T cells is unaffected by a Hexb deficiency [34].The authors did not however discuss the state of the thymus in later stages.In other LSDs, alterations in the last stage thymus have been previously reported in feline GM1 gangliosidosis [19,20].Flow cytometric analyses showed a marked decrease in the percentage of immature CD4 + /CD8 + T cells and a significant increase in CD4 2 /CD8 + cells in GM1 mutant cats of greater than 210 days of age when compared with normal age matched cats [19].Thymic involution has also been described in twitcher mice [21] and may therefore be a common pathological state among the LSDs.
We next examined for histological abnormalities in the thymus of Hexb 2/2 FcRc +/+ mice.In the 15 week old animals, the cellular density of the thymic cortices was found to be severely reduced as a result of a decreased number of CD4 + /CD8 + cells (Fig. 2).However, numerous macrophages with a ''foamy'' appearance were found instead in this region.Some of these macrophages contained hematoxylin-positive particles in their cytoplasm, suggesting that the population of apoptotic thymic cells was rapidly increased and/or that the macrophages had a diminished phagocytic capacity.Importantly, a pathology similar to that evident in mouse models appears to exist also in human SD (Fig. 3).Moreover, atrophy of the thymus has previously been described in an autopsy case report by Takahashi et al. [35], and in a personal communication from Tatematsu et al. [36].These observations suggest that thymic alterations occur in SD in both mouse and human via a common mechanism.
We next found in our mouse model that during thymic involution, nuclear particles in the cytoplasm of the macrophages, apoptotic thymic cells, and IgG deposition to the thymic cells were all increased, particularly in the cortex (Fig. 4).Most thymic lymphocytes are cleared by normal apoptotic processes but

Th1 cytokine
Tumor necrosis factor (Tnf) 14.1 50.4 1.9 Transforming growth factor, beta 1 (Tgfb1) 455 213 20.9 The AffymetrixH Mouse Genome 430 2.0 Array was employed to identify changes in the gene expression profile in the thymus of a 15 week old of Hexb 2/2 FcRc +/+ mouse.'Signal' represents a measure of the relative abundance of a particular transcript.'Exp/Base signal Log ratio' denotes the change in the expression level for a transcript between Base group (15 week old Hexb +/2 FcRc +/+ thymus) and the Experimental group (15 week old Hexb 2/2 FcRc +/+ thymus).This change is expressed as the log2 ratio.A log2 ratio of 1 is equivalent to a fold Change of 2. Representative genes that function in the immune system are listed.doi:10.1371/journal.pone.0012105.t001increased apoptosis can be observed in some animal models of LSD such as twitcher mice, and feline GM1 gangliosidosis.It is thought that the accumulation of GM2 induces neuronal cell death in GM2 gangliosidosis.Various extra-neural organs also contain many minor ganglioside components.In particular, the thymus has been found to have a very complex and characteristic spectrum of ganglioside species [37,38].Gadola et al have previously observed that glycolipids are stored in the thymus [22].In addition, Zhou et al. have reported that GM1, GM2, GM3, and GT1b can induce apoptosis in thymocytes in vitro [18].
We have also confirmed in our experiments that GM2 enhances thymic cell death, resulting in the formation of in vivo-like T cell subpopulations in vitro (data not shown).However, we also found that the apoptosis and necrosis of thymic cells is decreased in 15 week old Hexb 2/2 FcRc 2/2 mice, although the accumulation levels did not obviously differ from Hexb 2/2 FcRc +/+ mice (data not shown).This result suggests that the Fc receptor plays a role in thymic cell death in Hexb 2/2 FcRc +/+ mice.We have previously detected the presence of anti-GM2 and anti-GA2 autoantibodies in end stage Hexb 2/2 FcRc +/+ mice [10] and also other autoantibodies such as anti-ssDNA (unpublished data), which suggests the presence of polyclonal antibodies in Hexb 2/2 FcRc +/+ mice.In our present study, we observed the deposition of IgG on T cell membranes (Fig. 4E).These findings suggest that an anti-T cell autoantibody is produced during the last stages in Hexb 2/2 FcRc +/+ mice and that an IgG-FcR mediated pathway plays a role in the thymic involution seen in Hexb 2/2 FcRc +/+ mice.The thymus from Hexb 2/2 FcRc +/+ mice older than 15 weeks showed a marked increase in the percentage of macrophages, particularly in the cortex (Fig. 5A, B).Additionally, these macrophages were found to contain many nuclear particles in the cytoplasm.These results are consistent with the findings of previous histological experiments using H&E, TEM and TUNEL analyses.Mip-1a which is an important molecule in the pathogenesis of Hexb 2/2 mice [39], is upregulated in the thymus of 15 week old Hexb 2/2 FcRc +/+ mice.The evidence suggests therefore that the increase in the number of macrophages is partly due events that occur peripherally to the thymus.
To better understand the molecular basis of thymic involution, we performed cDNA microarray analysis to identify the changes in gene expression that accompanied involution in this gland.The results revealed an upregulation of immune response associated genes dominated by macrophages.A question that then emerged was how the macrophages were activated at this stage.It is very possible that enhanced cell death and/or the FcRc dependent signaling pathway is involved in the mechanism.Further analysis is needed to determine whether the FcRc pathway does contribute to gene expression changes in the thymus.On the other hand, prior to autoantibody production, FcRc independent pathways may induce thymic alterations.In this regard, previous reports have indicated that in the CNS of Hexb 2/2 mice, the accumulation of undegraded glycolipids in glial cells might be a trigger of pro-inflammatory cytokine/chemokine production [8,40].It has also been reported that the induction of Mip-1a might coincide with the accumulation of N-acetylhexosaminyl glycoconjugates due to a Hexb deficiency in glial cells [31].It is also known that macrophages accumulate considerable levels of glycolipids within late endosomes/lysosomes during the onset of Sandhoff disease [22].
CXCL13, an upregulated gene in end stage Hexb 2/2 FcRc +/+ mice, was found to be specifically expressed in the thymus, and B1 cells were also detected at increased levels in the thymus (Fig. 6A,  B, 7).High expression of CXCL13 has been found previously in many autoimmune diseases, such as myasthenia gravis [31], multiple sclerosis [33], and in a model mouse of SLE [26], and is considered to be associated with autoantibody production.We have shown also in our previous study that a significant elevation of serum antibody levels occurs in the terminal stages of Hexb 2/2 FcRc +/+ mice of more than 14 weeks of age [10].Thus, a high expression of CXCL13 and infiltration of B1 cells in Hexb 2/2 FcRc +/+ mice may be a reflection of autoantibody production.
Based on our current results, we propose a mechanism by which thymic alterations occur in Hexb deficient mice as follows.The apoptosis of immature T cells occurs normally in Hexb 2/2 FcRc +/+ mice and these apoptotic T cells are engulfed by macrophages.However, macrophages cannot degrade the glycolipids from apoptotic T cells in these animals because of their Hexb deficiency, and thus accumulate these molecules in lysosomes.The macrophages are then activated and produce CXCL13, which promotes chemotaxis toward B1 cells and thus leads to the development of autoimmunity.Once autoantibodies against T cells are produced, the storage of T cell-derived glycolipids by macrophages is enhanced via the autoantibody-dependent phagocytosis of T cells.This undesired loop results in a disrupted immune system and in the production of autoantibodies against neuronal cell antigens or neuronal cell -T cell common antigens such as GA2.Deletion of FcRc may thus prevent thymic involution by blocking the autoantibody dependent phagocytosis of T cells and suppressing specific gene expression.A reduction of the serum titer against GM2 and GA2 in Hexb 2/2 FcRc 2/2 mice [10] might be related to thymic integrity.Further studies involving other factors, such as functional testing of single lymphocytes and innate immunity, will be needed to further our understanding of autoimmunity in GM2 gangliosidoses.

Human thymus
A hematoxylin and eosin (H&E) stained slide of a human thymus, dissected from an 11-month-old male with SD who was autopsied at Kobe Children's Hospital [47], was kindly loaned by Dr. H. Itoh.

cDNA microarray
Pooled thymic RNA samples were obtained from two 15 weeks old Hexb 2/2 mice and two strain-matched, 15 weeks old Hexb +/2 mice.cDNA were synthesized by GeneChip T7-Oligo(dT) Promoter Primer Kit (Affymetrix, Inc, Santa Clara, CA) and TaKaRa cDNA Synthesis Kit (TaKaRa Bio Inc, Shiga, Japan) from 10 mg total RNA.Biotinylated cRNA were synthesized by IVT Labeling Kit (Affymetrix).Following fragmentation, 10 mg of cRNA were hybridized for 16 hr at 45uC on GeneChip Mouse Genome 430 2.0 Array (Affymetrix) containing 45101 unique mouse genes.GeneChips were washed and stained in the Affymetrix Fluidics Station 450.GeneChips were scanned using GeneChip Scanner 3000 7G.Single Array Analysis were calculated by Microarray Suite version 5.0 (MAS5.0)with Affymetrix default setting and global scaling as normalization method.The trimmed mean target intensity of each array was arbitrarily set to 500.Signals were calculated using the One-Step Tukey's Biweight Estimate which yields a robust weighted mean.The signal log ratio was computed using a one-step Tukey's Biweight method by taking a mean of the log ratios of the probe pair intensities across the two arrays.All data is MIAME compliant and that the raw data has been deposited in GEO.

Real-time RT-PCR
Total RNA was purified with TRIzol Reagent (Invitrogen).To determine the high CXCL13-expressing cells, a magnetic cell sorting system was used for the RNA preparation.Briefly, a 1610 6 thymic cell suspension from 15 week old Hexb 2/2 FcRc +/+ mice was stained with CD11b MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and separated using an MS Column (Miltenyi Biotec), and a MiniMACSH Separator (Miltenyi Biotec).cDNA was synthesized from total RNA by reverse transcription using a commercial cDNA synthesis kit (TaKaRa Bio Inc).cDNA synthesized from 500 ng of total RNA was used as the template in each reaction.The relative gene expression levels were determined using the SYBRH Premix Ex Taq TM II (TaKaRa Bio Inc).The primer sets for ribosomal protein s18 (Rps18) and tumor necrosis factor-alpha (TNF-a) were obtained from TaKaRa Bio.The primer set for macrophage inflammatory protein-1 alpha (Mip-1a) has been described previously [31].The primers used to amplify Chemokine (C-X-C motif) ligand 13 (CXCL13) are 59-TCTCTCCAGGCCACGGTATTCT-39 (forward, F) and, 59-ACCATTTGGCACGAGGATTCAC (reverse, R) and for stromal cell-derived factor 1 (SDF-1) are 59-GAGCCAACGTCAAG-CATCTG-39 (F) and 59-CGGGTCAATGCACACTTGTC-39 (R).Rps18 was amplified in each reaction simultaneously as a standard control.The fluorescence changes from each well were monitored using an ABI PRISMH 7500 Sequence Detection System (Perkin Elmer, Inc, Waltham, MA).

Statistical analysis
Statistical analysis was performed using the Student's t test.A 95% confidence limit was taken as significant.