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NDT Advance Access originally published online on April 23, 2007
Nephrology Dialysis Transplantation 2007 22(9):2540-2548; doi:10.1093/ndt/gfm228
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

T-cell homing receptor expression in IgA nephropathy

Arvind Batra, Alice C. Smith, John Feehally and Jonathan Barratt

Department of Infection, Immunity and Inflammation, University of Leicester, John Walls Renal Unit, Leicester General Hospital, Leicester LE5 4PW, UK

Correspondence and offprint requests to: Dr Jonathan Barratt, John Walls Renal Unit, Leicester General Hospital, Leicester LE5 4PW, UK. Email: jb81{at}le.ac.uk



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA (pIgA). In IgAN, mucosal pIgA production is reduced while systemic production is increased, making the latter the likely source of mesangial pIgA, and suggesting a displacement of pIgA-producing cells from mucosal to systemic sites. Upon activation, lymphocytes migrate through the circulation up-regulating homing receptors (HR) which direct their return to appropriate effector locations. We investigated the HR expression of T-cell subsets in IgAN, healthy adults and membranous nephropathy (MN).

Methods. Peripheral blood cells were labelled for CD3, CD4 and CD8, and for L-selectin (naive cells), integrin {alpha}4ß1 (systemically homing cells) and integrin {alpha}4ß7 (mucosally homing cells) and analysed by flow immunocytometry.

Results. In IgAN, CD3 T cells displayed reduced L-selectin and increased {alpha}4ß1hi expression, with no difference in {alpha}4ß7. No abnormality of T-cell HR expression was found in MN. Both IgAN and healthy adults maintained their patterns of T-cell HR expression when studied again at a later time point, and the changes in IgAN were entirely accounted for by the CD4 T-cell subset with CD8 HR expression being normal.

Conclusions. The consistently reduced L-selectin expression by CD4 T cells indicates increased activation of this subset in IgAN. These activated cells express {alpha}4ß1 rather than {alpha}4ß7, and therefore home to systemic effector sites. CD4 T cells regulate antibody production, including IgA. As pIgA is overproduced in systemic sites in IgAN, we hypothesize that these activated systemic homing CD4 T cells may direct the aberrant systemic pIgA production observed in IgAN.

Keywords: IgA nephropathy; {alpha}-4 integrins; lymphocyte homing; pathogenesis; T cell



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
IgA nephropathy (IgAN) is one of the commonest patterns of glomerulonephritis (GN), and is characterized by deposition of polymeric IgA (pIgA) in the renal mesangium. A number of subtle disturbances of the IgA immune system have been reported in IgAN [1], which together lead to the presence in the blood of pIgA molecules with a propensity to mesangial deposition, although the mechanisms underlying this phenomenon have yet to be fully elucidated.

Human IgA production occurs in two distinct immunological compartments: mucosal and systemic. The vast majority of IgA is produced at mucosal surfaces. This is almost exclusively polymeric and is transported directly across the mucosal epithelium into external secretions, with very little entering the blood. Serum IgA, which is mostly monomeric (mIgA), arises from the systemic immune compartment and is mainly produced in the bone marrow. Therefore, pIgA can be considered to be the mucosal IgA phenotype. The factors controlling pIgA production rather than monomeric IgA (mIgA) in mucosal immune tissue are unknown, but it is clear that lymphocytes encountering antigen at mucosal sites are at some point subjected to influences which strongly favour pIgA production over that of mIgA and other immunoglobulin isotypes.

There is good evidence for disturbances in lymphocyte populations in patients with IgAN. The numbers of pIgA-producing plasma cells are reduced in the duodenal mucosa of patients with IgAN [2], but increased in the bone marrow [3,4], arguing against overproduction of mucosal pIgA, but rather suggesting a systemic source for mesangial pIgA. Immunization studies show exaggerated systemic pIgA responses to both systemic and mucosal antigens [5,6], while some mucosal pIgA responses to mucosal antigen challenge are reduced in IgAN [7,8]. Furthermore, elevated circulating pIgA antibody levels have been described against mucosal antigens such as polio, Helicobacter pylori, and food and environmental components in IgAN [9,10]. Therefore, although the circulating pIgA in IgAN is likely to arise from a systemic source, it may well be driven by mucosally encountered antigens. Overall, this picture strongly suggests a shift of mucosal-type antibody production from mucosal to systemic sites, and indicates that abnormal control of the fine balance between the mucosal and systemic immune systems may underlie the pathogenesis of IgAN.

Restriction of cell- and antibody-mediated immune responses to appropriate sites in the body is achieved by the interactions of specialized adhesion molecules, or homing receptors (HRs), expressed on the surface of B and T lymphocytes with vascular endothelial ligands. The expression of lymphocyte HRs varies with the activation status of the cell, and naïve, effector and memory lymphocytes display different homing characteristics [11]. The main HR of naïve lymphocytes is L-selectin (CD62L). L-selectin can bind both the peripheral lymph node adhesion molecule (PNAd) and the mucosal addressin cell adhesion molecule MAdCAM-1, and therefore, mediates the migration of naïve lymphocytes through both systemic and mucosal lymphatic tissues [12]. If antigen is encountered in these induction sites, lymphocytes differentiate into short-lived effector T cells and a minor fraction of long-lived memory T cells and migrate through the circulation before homing back to the site of antigen encounter to elicit immune responses appropriate to the location, for example to direct, the dominant production of pIgA antibodies at mucosal sites.

T cells are pivotal in the control of antigen-driven adaptive immune responses, and it is likely that mucosally activated T cells play an important role in driving mucosal antibody production towards a pIgA bias. Effector T lymphocytes (CD4 and CD8) downregulate CD62L [13], whilst up-regulating expression of {alpha}4-integrin HRs dependent upon the site of activation [14]. The best characterized integrin HRs are {alpha}4ß1 (also known as VLA-4) and {alpha}4ß7 [15]. The ligand for {alpha}4ß1 is vascular cell adhesion molecule 1 (VCAM-1) [16], the expression of which is up-regulated at non-mucosal sites of inflammation [17]. Therefore, T cells expressing {alpha}4ß1 preferentially home to systemic sites including the skin, nervous system and bone marrow. The {alpha}4ß7 binds the mucosal addressin cell adhesion molecule MAdCAM-1, and therefore, {alpha}4ß7 expressing T cells preferentially migrate to mucosal sites [18]. Lymphocyte up-regulation of ß1 and ß7 is mutually exclusive, and therefore, in the circulation, these integrins define T cells homing to systemic and mucosal sites respectively [19].

We hypothesize that altered homing of mucosally-activated T cells may underlie the aberrant production of pIgA observed in IgAN. To address this possibility, we investigated the expression of CD62L and the integrins {alpha}4ß1 and {alpha}4ß7 on peripheral blood T-cell subsets from patients with IgAN, healthy adults and patients with membranous nephropathy (MN). In IgAN, we found an increased percentage of CD4 T cells expressing the systemic HR {alpha}4ß1, paralleled by a decrease in CD62L expression, indicating a higher proportion of systemically homing activated effector and CD4 T cells in IgAN.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgements
 References
 
Materials
Unless otherwise stated, all reagents and materials were purchased from Sigma Chemical Co. (Poole, Dorset, UK). All antibodies were purchased from Pharmingen (BD Biosciences, Cowley, Oxford, UK), except anti-integrin ß1 (anti-CD29, Serotec, Kidlington, Oxford, UK), and anti-integrin {alpha}4ß7 (Act-1, previously described by Schweighoffer [18]) which was a kind gift from Millenium Pharmaceuticals Inc (Cambridge, MA, USA).

Subjects
We studied 38 patients with biopsy-proven IgAN (22 male, mean age 38 years, range 20–57), 46 healthy adults with no history of kidney disease (23 male, mean age 34 years, range 21–57), and 7 patients with biopsy-proven idiopathic membranous nephropathy (MN) (5 male, mean age 44 years, range 28–56). 12 randomly selected IgAN patients (6 male, mean age 34 years, range 20–51) and 11 healthy adults (6 male, mean age 35 years, range 25–50) were studied a second time, a minimum of 3 months after their initial recruitment. All subjects gave informed consent for inclusion in the study.

At the time of the study and during the follow-up period, no subject had an intercurrent illness, and none of the IgAN patients had macroscopic haematuria. The median time from renal biopsy to entry into the study for IgAN patients was 5 years (range 0.5–8). All IgAN patients had microscopic haematuria and/or proteinuria, but proteinuria was <1.5 g/24 h in all cases. All patients with idiopathic MN had <1.5 g/24 h proteinuria. The median serum creatinine in the IgAN patients was 117 µmol/l (range 65–285), and in MN was 104 µmol/l (range 70–170). None of the patients or healthy adults was receiving immunosuppressive treatments at the time of the study or had received treatment in the 24 months prior to study entry.

Peripheral blood mononuclear cell (PBMC) preparation
PBMCs were isolated from heparinized venous blood samples by density gradient centrifugation on Histopaque 1077 for 30 min at room temperature at 400 g. Cells were washed twice in ice cold PBS/2mM EDTA/0.5% BSA (FACS buffer), resuspended in the same buffer at 1 x 107/ml, and immediately used for staining.

Flow immunocytometry
Cells were labelled with specific combinations of directly fluorochrome-conjugated or unconjugated antibodies. Where unconjugated primary antibodies were used, cells were sequentially labelled with unconjugated antibody followed by species-specific fluorochrome-conjugated secondary antibody. Non-specific and Fc-mediated binding was minimized by using F(ab)2 conjugates and blocking with normal species-specific sera before staining. Samples were washed in ice cold FACS buffer between each step, all steps were performed on ice and following labelling, cells were immediately analysed by three colour flow immunocytometry.

Lymphocyte subsets were identified by staining with fluorochrome-labelled antibodies against CD19 (B cells), CD3 (T cells), CD4 (helper T cells) and CD8 (cytotoxic T cells). HR expression was assessed by simultaneous staining with antibodies to L-selectin (CD62L), integrins {alpha}4 (CD49d) and ß1 (CD29), or the {alpha}4ß7 heterodimer.

Appropriate negative controls were prepared by incubating cells with fluorochrome-labelled isotype control antibodies. Propidium iodide (PI) staining was used to exclude dead cells from all analyses.

All data were acquired and analysed with an FACScan flow cytometer coupled to CELLQuest software (Becton Dickinson, San Jose, CA, USA). Lymphocytes were selected according to forward and side scatter parameters, dead cells were excluded and a minimum of 104 cells analysed. For each fluorescence channel appropriate negative isotype controls were run with each sample series and the percentage of cells staining positive with each antibody calculated. Over the duration of the experiments, the FACScan was calibrated weekly for fluorescence and light scatter with microbead standards (Dako FluoroSpheres, Dako Ltd, High Wycombe, Bucks, UK).

Analysis of {alpha}4ß1 integrin expression
There is no single antibody available that directly reacts with the whole {alpha}4ß1 heterodimer, and therefore, it was necessary to carry out double-staining with antibodies directed against the {alpha}4 and ß1 chains individually to identify cells with the systemic homing phenotype. In all subjects studied, upwards of 50% of CD3 T cells were positive for ß1 integrin expression but, as described by others, these positive cells fell clearly into two distinct populations with high and low ß1 expression (Figure 1) [19,20]. As expected, identical staining patterns were found when gating on CD4 and CD8 T-cell subsets. Therefore, during flow immunocytometric analysis we calculated the total percentage of cells co-expressing {alpha}4 and ß1 integrins, the percentage expressing {alpha}4 with Formula , and also the percentage of ß1-positive cells that fell into the Formula subpopulation. We consistently found that >95% of Formula T cells co-expressed {alpha}4, and therefore only present data here on those lymphocytes displaying a Formula phenotype, as these lymphocytes represent the activated effector and memory T cells, primed for homing to non-mucosal sites.


Figure 1
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  		Fig. 1. Flow immunocytometric analysis of integrin ß1 expression by T lymphocytes. In all subjects, staining of CD3 lymphocytes for ß1 expression displayed a clear biphasic distribution. The Figure 1 phenotype, defined as the right-hand peak on the histograms, defines effector and memory T cells homing to non-mucosal sites. Compared with healthy adults, T cells from patients with IgAN showed a significant increase in the proportion of cells displaying a Figure 1 phenotype, indicating an increase in the population of systemic homing T cells. The same biphasic distribution of ß1 staining was seen in both CD4 (A) and CD8 (B) T-cell subsets, although the increase in Figure 1 in IgAN only reached significance for the CD4 subset.

 
Statistical analysis
Data analysis was by Mann–Whitney U tests, ANOVA, regression analysis and paired t-tests. P values <0.05 were considered significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgements
 References
 
Lymphocyte subsets in IgAN
Consistent with other studies, no difference was found between patients with IgAN and healthy adults in the distribution of the main lymphocyte subsets: CD3: CD19 (healthy adults 4.9 ± 0.5, IgAN 4.6 ± 0.6); CD4: CD8 (healthy adults 2.4 ± 0.2, IgAN 2.2 ± 0.2); P > 0.05 in all cases.

CD3 T-cell homing receptor expression
In an initial study of 38 patients with IgAN and 46 healthy adults, there was no difference between the subject groups in the percentage of Formula CD3 T cells (healthy adults: 48 ± 2.6%; IgAN: 50.5 ± 2.4%), or in the intensity of {alpha}4ß7 staining in the Formula T-cell subset (data not shown). However, we noted a trend towards a reduced percentage of CD62Lhi CD3 T cells in IgAN (healthy adults: 79.8 ± 1.5%; IgAN: 73.7 ± 2.4%; P = 0.054), and a significant increase in the percentage of Formula CD3 T cells (the systemic homing phenotype) in IgAN (healthy adults: 40.9 ± 1.9%, IgAN: 46.9 ± 2.1%, P = 0.034) (Figure 2). Commensurate with this, the percentage of {alpha}4ß1-positive cells that fell into the ß1hi range of the biphasic distribution was increased in IgAN (healthy adults 53.4 ± 2.1%, IgAN 59.7 ± 2.2%, P = 0.03). There was no difference between healthy adults and IgAN in the intensity of CD62L staining in the CD62Lhi T-cell subset (data not shown). There was also a significant negative correlation between the percentage of CD62Lhi CD3 T cells and Formula CD3 T cells in IgAN (Figure 3), demonstrating the expected inverse relationship between CD62L, the dominant HR of naïve T cells, and ß1 integrin, which is up-regulated following lymphocyte encounter with antigen. Finally, there was no correlation between the expression of any homing receptor and the degree of renal impairment (eGFR calculated by the modified MDRD formula) or the rate of urinary protein excretion (protein creatinine ratio) at the time of venous sampling. This was confirmed when data from the second study (as follows) were included incorporating both IgAN and MN patients (data not shown).


Figure 2
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  		Fig. 2. Homing receptor expression by peripheral blood T cells in IgA nephropathy and healthy adults. Flow immunocytometric analysis of lymphocytes double stained for CD3 and CD62L, {alpha}4ß1 or {alpha}4ß7. Compared with healthy adults (open bars; n = 46), we found in patients with IgAN (closed bars; n = 38) a trend to a lower percentage of CD62Lhi CD3 cells, and a significantly increased percentage of Figure 2 CD3 cells. The percentage of Figure 2 CD3 cells was not different across the subject groups.

 

Figure 3
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  		Fig. 3. Relationship between CD62Lhi and Figure 3 T cells in peripheral blood in IgAN. The percentages of circulating CD62Lhi CD3 T cells and Figure 3 CD3 T cells showed a significant negative correlation.

 
These observations suggest that compared with healthy adults, in IgAN, a higher percentage of circulating T cells are activated T cells, and that these are more likely to home to systemic than mucosal effector sites.

Homing receptor expression by T-cell subsets
In order to confirm our initial observations and further characterize the phenotype of the increased systemic homing CD3 T cell population in IgAN, we re-studied 23 subjects at a second time point, not less than 3 months later (12 IgAN, 11 healthy adults). In this study, we additionally included seven patients with idiopathic MN, to establish whether abnormal T-cell homing is specific to patients with IgAN, or is also seen in other forms of glomerular disease. In this second study, we analysed the HR expression of CD4 and CD8 T-cell subsets separately.

Comparison of homing receptor expression by CD4 and CD8 T cells
Consistent with previous reports in healthy adults, a greater proportion of CD4 T cells expressed CD62L than CD8 T cells (CD4 92.8 ± 1.7%, CD8 72.0 ± 2.7%, P < 0.001). In contrast, there was no difference in the percentage of CD4 and CD8 T cells expressing {alpha}4ß1, or in the distribution of Formula staining (Table 1), while the mucosal HR {alpha}4ß7 was preferentially expressed on CD8 T cells (CD4 41.7 ± 3.2%, CD8 56.9 ± 5.2%, P = 0.017).


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  		Table 1. Comparison of homing receptor expression by CD4 and CD8 T-cell subsets in healthy adults, IgA nephropathy and membranous nephropathy

 
Homing receptor expression by CD4 T cells in patients and healthy adults
In IgAN, the percentage of CD62Lhi CD4 T cells was lower than healthy adults (healthy adults 92.8 ± 1.7%, IgAN 84.9 ± 2.6%, P < 0.01), while Formula expression was higher (healthy adults 43.2 ± 3.1%, IgAN 56.7 ± 3.8%, P < 0.01). There was no difference between healthy adults and IgAN in the intensity of CD62L staining in the CD62Lhi T-cell subset (data not shown). As before, the increased frequency of cells expressing Formula was associated with a shift in the distribution of ß1 staining (percentage of total ß1 positive CD4 cells falling into the Formula subpopulation: healthy adults 60.1 ± 2.0%, IgAN 70.3 ± 2.2%, P < 0.001). There was no difference in either the percentage of Formula CD4 T cells or in the intensity of {alpha}4ß7 staining in the Formula CD4 T-cell subset between IgAN and healthy adults. HR expression by CD4 T cells from patients with MN did not differ significantly from healthy adults (Figure 4).


Figure 4
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  		Fig. 4. Expression of homing receptors by CD4 T cells in IgAN. Flow immunocytometric analysis of lymphocytes double stained for CD4 and CD62L, {alpha}4ß1 or {alpha}4ß7. Compared with healthy adults (n = 11, open bars) and membranous nephropathy (n = 7, hatched bars), in IgAN (n = 12, closed bars), there was a significantly lower percentage of CD62Lhi CD4 T cells, and a higher percentage of Figure 4 CD4 T cells. CD4 T cell expression of {alpha}4ß7 was not different in IgAN and healthy adults, and patients with MN did not differ from healthy adults in CD4 T cell expression of any homing receptor.

 
Homing receptor expression by CD8 T cells in patients and healthy adults
In contrast, patients with IgAN did not display any significant alteration in the percentage of CD8 T cells with a CD62Lhi, Formula or Formula phenotype when compared with healthy adults or MN (Figure 5).


Figure 5
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  		Fig. 5. Expression of homing receptors by CD8 T cells in IgAN. Flow immunocytometric analysis of lymphocytes double stained for CD8 and CD62L, {alpha}4ß1 or {alpha}4ß7. There was no difference in the percentage of CD62Lhi, Figure 5 or Figure 5 CD8 T-cell subsets between patients with IgAN (n = 12, closed bars), healthy adults (n = 11, open bars) and patients with MN (n = 7, hatched bars).

 
Influence of time on T-cell homing receptor expression
To establish whether the observed pattern of T cell HR expression was persistent over time, we compared the results obtained from 12 patients and 11 healthy adults who participated in both studies at two different time points, T1 and T2, which were a minimum of 3 months apart. Individual subjects showed remarkable consistency in their T- cell HR expression over time, with a lower percentage of CD62Lhi and higher percentage of Formula T cells being apparent in the IgAN group at both time points (Figures 6 and 7). The proportion of Formula T cells equally did not vary across the two time points and remained unchanged from healthy adults.


Figure 6
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  		Fig. 6. CD62L expression by CD3 T cells at different time points. In 12 patients with IgAN (closed circles) and 11 healthy adults (open circles), the expression of CD62L by CD3 T cells was analysed by flow immunocytometry at two time points not <3 months apart. The percentage of CD62Lhi CD3 T cells did not change significantly over this time, and the previously observed reduction in CD62Lhi CD3 T cells in patients with IgAN was persistent.

 

Figure 7
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  		Fig. 7. {alpha}4ß1 expression by CD3 T cells at different time points. In 12 patients with IgAN (closed circles) and 11 healthy adults (open circles) the expression of {alpha}4ß1 by CD3 T cells was analysed by flow immunocytometry at two time points not <3 months apart. The percentage of Figure 7 CD3 T cells did not change significantly over this time, and patients with IgAN retained a raised percentage of Figure 7 CD3 T cells.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgements
 References
 
In this study, we investigated the possibility that alterations in T-cell homing may be involved in the aberrant pattern of mucosal-type pIgA1 production seen in patients with IgAN. We found a persistent increase in the percentage of CD4 T cells expressing {alpha}4ß1, which favours their recruitment to non-mucosal sites, and a corresponding decrease in expression of CD62L by CD4 T cells in IgAN. However, in IgAN we found no changes in mucosal homing T-cell populations, characterized by surface expression of the alternative {alpha}4 integrin {alpha}4ß7. Taken together, these findings indicate a higher percentage of circulating T cells are activated effector cells in IgAN and that these T cells are more likely to home to systemic than mucosal effector sites.

L-selectin is uniformly expressed by naïve circulating T lymphocytes and mediates T-cell recruitment into peripheral lymph nodes, but not into extra-lymphoid tissue [21]. It is well-recognized that lymphocyte expression of the integrin chains {alpha}4, ß1 and ß7 are variable, leading to T-cell subsets with high and low expression [20,22]. Antigen encounter and T-cell activation are marked by up-regulation of {alpha}4 along with one of these two ß chains, leading to distinct populations of effector and memory T cells ex-pressing Formula or Formula with the capacity to traffic through non-lymphoid tissues. Expression of Formula is the hallmark of gut-homing lymphocytes enabling these cells to attach to mucosal MAdCAM-1, while Formula (VLA-4) preferentially binds to VCAM-1 and fibronectin, and thereby recruits cells systemically to areas of activated endothelium or exposed extracellular matrix. Co-expression of CD62L with {alpha}4 integrins appears to bias T-cell recirculation to trafficking through lymphoid tissues while CD62Llo/– T cells display a relative preference for entering extralymphoid sites [19]. The factors controlling the type of integrin HR expressed by activated T cells are not well understood, although evidence is accumulating for the critical role of dendritic cells [23]. Furthermore, it is as yet unclear whether site-specific integrin expression is achieved by up-regulaton of appropriate receptors by T cells equally able to express all HR, or preferential ‘selection’ and clonal expansion of T cells pre-committed to express specific integrin chains [24].

In all the subjects examined, the CD4 and CD8 T-cell subsets demonstrated consistent patterns of HR expression. As previously demonstrated, the CD8 subset had lower CD62L and increased {alpha}4ß7 expression when compared with CD4 T cells [25]. We found no difference in the percentage of circulating CD4 or CD8 cells expressing Formula or in the intensity of {alpha}4ß7 staining between patients and healthy adults. The gut mucosa is the major site of IgA production in man, and there is good evidence for disrupted IgA production in IgAN, with reduced numbers of J chain and IgA1-producing plasma cells and compromised mucosal IgA1 antibody responses to mucosal antigen challenge [2,7]. Our study does not provide any evidence for altered mucosal-homing T-cell populations playing a direct role in reduced mucosal pIgA1 production in IgAN. However, the {alpha}4ß7-expressing CD4 and CD8 T cells identified in this study will have been predominantly memory T cells. We cannot, therefore, exclude a selective defect in Formula expression in effector T cells in IgAN. Furthermore, mucosal homing is not mediated exclusively by {alpha}4ß7 and there is increasing evidence to support the importance of a number of chemokine receptors such as CCR9 and CCR10 in lymphocyte trafficking to mucosal epithelial sites [26]. These were not assessed in this particular study, but are the focus of a separate investigation. Equally, we cannot exclude subtle imbalances in either the antigen specificities or functional characteristics of the {alpha}4ß7 T-cell population we have detected. Also, effective homing of these cells back to appropriate mucosal sites depends on additional local factors such as expression of MAdCAM-1 and synthesis of chemokines such as MEC (mucosae-associated epithelial chemokine/CCL28) and TECK (thymus-expressed chemokine/CCL25) [27]. Few studies have investigated mucosal T-cell populations in situ in IgAN. Increased T-cell activation marker expression and altered V region usage by mucosal {gamma}{delta} T cells have been reported [28,29], indicating that there is, in fact, some disruption of mucosal T-cell function, but the significance of these findings is as yet unclear.

Our major finding in IgAN was an increase in the percentage of circulating CD4 T cells co-expressing high levels of the integrin {alpha}4 and ß1 chains, the components of the HR heterodimer {alpha}4ß1 (VLA-4). We also found a reduced proportion of CD62Lhi CD4 T cells in IgAN, which showed a significant negative correlation with Formula . Taken together, this suggests that in IgAN there are increased numbers of CD4 T cells capable of homing to non-mucosal sites. As CD62L is often downregulated by antigen-experienced T cells, this is probably a further reflection of increased CD4 activation in this disease [30].

These observations contradict to some extent earlier findings suggesting increased CD62L expression by T cells in IgAN [31,32]. One of these studies reports on lymphocyte HR profiles in children with IgAN, and therefore, it is difficult to compare with adult data as HR expression is known to change with age [33]. The study by Kennel-De March reported increased CD62L density on T lymphocytes in IgAN (unconfirmed in this study). However, consistent with our findings, this study could not demonstrate a significant difference in the proportion of CD62Lhi T cells when comparing healthy adults and IgAN subjects. This earlier study examined enriched T and B lymphocytes rather than whole blood and it may be that this relatively prolonged experimental protocol led to changes in the surface phenotype of the lymphocytes and the subtle increase in antigen staining reported. With regard to T cell ß1 expression, we have specifically evaluated the Formula subset of the bimodal ß1 staining profile. Comparison of the percentage of {alpha}4ß1-staining T cells in this study (48.0–50.5%) with that seen in the Kennel-De March study (71.3–83.5%) would suggest that the criteria used to define Formula T cells differ considerably making comparisons impossible. Importantly, this earlier study did not examine HR expression in T-cell subsets, and therefore was unable to comment on changes in CD4 CD62L and {alpha}4ß1 expression. Acknowledging the discrepancy between our findings and earlier reports we randomly selected a proportion of the healthy adults and IgAN subjects and repeated their HR staining. This did not differ significantly across the two time points suggesting that our initial observations were indeed correct. This stability is perhaps not surprising as we were predominantly studying the phenotypes of naïve and memory T-cell populations, which would not be expected to change significantly in the absence of acute infection or inflammation.

We can only speculate as to the likely destination of Formula CD4 T cells in IgAN. The main ligand of {alpha}4ß1 is VCAM-1 [34,35]. In contrast to the {alpha}4ß7 ligand MAdCAM-1, which is restricted to mucosal venules where it is constitutively expressed, VCAM-1 can be induced in the vasculature of a wide variety of tissues in inflammatory conditions, but is rarely expressed in the mucosa, even when inflamed [19,36]. VCAM-1, therefore, mediates recruitment of Formula cells to inflamed non-mucosal sites [37–39]. In IgAN there is an increase in pIgA1 antibody production at systemic sites such as the bone marrow [3,4]. Interestingly, there is good evidence for a key role for VCAM-1/Formula interactions in normal cellular recruitment to the bone marrow [40–42]. We would suggest that the increase in systemically homing CD4 T cells in IgAN includes a subset of helper T cells capable of supporting a mucosal-type, pIgA-dominated immune response at non-mucosal sites such as the bone marrow. It is our belief that the continual release of systemically produced pIgA into the blood, perhaps under the control of systemically homing T helper cells, overwhelms normal clearance mechanisms and, along with physical characteristics predisposing to mesangial deposition, results in the glomerular pIgA1 accumulation pathognomonic of IgAN. In order to formally test this hypothesis, we are now assessing antigen specific T- and B-cell homing patterns and effector phenotypes during immune responses to mucosally and systemically administered neo- and recall antigens.



   Acknowledgements
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
References
 
A.B. was supported with a grant from the National Kidney Research Fund (R38/1/2000) and J.B. holds a DoH Clinician Scientist Award. We would like to thank Millenium Pharmaceuticals Inc., Cambridge, MA, USA for the kind gift of their antibody Act-1.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Barratt J, Feehally J, Smith AC. Pathogenesis of IgA nephropathy. Semin Nephrol (2004) 24:197–217.[CrossRef][Web of Science][Medline]
  2. Harper SJ, Pringle JH, Wicks AC, et al. Expression of J chain mRNA in duodenal IgA plasma cells in IgA nephropathy. Kidney Int (1994) 45:836–844.[Web of Science][Medline]
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Received for publication: 19.12.06
Accepted in revised form: 23. 3.07


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