5.2.4B Cell Monoclonal Expansion

Virtually all patients meeting the classification criteria for SjS [67] present with an association between salivary gland lymphocytes foci and ductal epithelial cell proliferation designated as lymphoproliferative sialadenitis. Straightaway, the question arises as to the relevance of such B cell aggregates to the pathophysiology of the syndrome. There are phenotypic findings to support that the salivary gland clusters of B cells are benign [68]. The issue turns as to why lymphoma develops at high frequency on the background of polyclonal proliferation of B cells in patients with SjS [49]. This issue was first addressed by Bunim and Talal who noticed that malignant lymphoma occurred in a patient with primary SjS [69]. Then, Talal and Bunim reported on the association of these two B-cell aberrations [70], and showed that the whole sequence from benign to malignant B proliferations was represented in the patients with primary SjS, but not in those with secondary SjS [71].

This seminal observation changes the view of lymphoma from an all-or-none phenomenon to one of a continuous spectrum of disease [72]. Ensuing studies revealed that widespread deregulation of lymphoid tissue complicates SjS. The clinical course and the evolution of patients with primary SjS and non-Hodgkin’s lymphoma (NHL) has underlined the frequency of MZ [73] lymphoma, and extranodal manifestations [74]. Intriguingly, extra-salivary lymphoma may supersede the original autoimmune setting. The long duration of sicca symptoms and the appearance of benign salivary gland lesions indicate that SjS is the first to develop. Such findings highlight the importance to delineate the order of events leading to uncontrollable B-cell transformation. The presence of palpable purpura and low C4 levels at the first visit distinguishes the patients at risk of developing NHL from those with uncomplicated disease [75]. Neutropenia and cryoglobulinemia are also associated with an increased incidence of NHL [76], and RF-producing B lymphocytes are frequently involved in such proliferation [77]. Whatever it is, B cells have come under suspicion as the lymphocyte population that initiates the entire pathological process.

5.3B Cells Are Not Dispensable

5.3.1B Cell Chemokines and Antibody Production

Three pieces of evidence deserve now to be discussed to highlight the role of B lymphocytes. The first is the constitutively expressed but functionally impaired chemokine receptor CXCR3 for T cells on epithelial cells [78], coupled with the aberrantly expressed B-cell-attracting chemokine CXCL13 on epithelial cells [79], and lymphoid infiltrates [80] recruit SjS-related B cells expressing CXCR5 into the salivary glands [81]. Organ-specific inflammation might develop through the aberrant expression, of a single chemokine within a given tissue. This process seems, however, to be regulated by the accumulation of a certain mass of cells and the subsequent

Sphingosine1P

production of lymphotoxins (LT). A particularly important function of B cells may be the effect they have on lymphoid neogenesis through the production of LT.

The second evidence for B cells is that certain autoantibodies encountered in this disease are pathogenic. An appealing example is the antimuscarinic receptor antibody that inhibits the mechanism of fluid secretion by human submandibular salivary acinar cells [82], and further alters the stimulated secretion of saliva when infused into nonobese diabetic Ig mnull mice [83]. Similar pathogenic agents are, probably, anticarbonic anhydrase II [84], and, possibly anti-a-fodrin autoantibodies [85].

The third evidence is that treatment of primary SjS with a monoclonal antibody directed against CD20 has proved effective in depleting B cells from the blood [86] and the salivary glands [87] of these patients, and in relieving their complaints (reviewed in [88]).

5.3.2Peculiarities of B Cell Products: Cytokines and IgA Autoantibodies

Three additional observations support aberrant B-cell function in SjS. Discovery of BAFF was not a tangential development, because BAFF is sufficient to induce a SjSlike disease with an expansion of BT2 and MZ B cells in the salivary glands of the mice [30]. Interestingly, we found similar features in B cells eluted from human salivary glands [21, 59]. Due to sphingosine 1-phosphate (see Fig. 5.4) inserted into its receptor, these MZ B cells might resist the CXCL13-mediated attraction toward the germinal centers [89]. The presence of this receptor on the surface of inflammatory mononuclear cells [90] facilitates the infiltration of these MZ B cells, where the receptor occupancy prevents their migration under the effect of CXCL13 [91]. IL-7 has also been suspected to favor the survival of lymphoid tissue B cells [92]. These findings do not prove that MZ B lymphocytes are pathogenic in SjS, but open up this possibly.

It is also relevant to the dysfunction of B lymphocytes that serum IgA are heavily glycosylated in primary SjS [93], owing to an excessive activity of a2-3- and

a2-6-sialyl-transferases in B lymphocytes [94]. As a result, IgA with overexpression of sialic acid in both their Fab and Fc regions are not recognized by asialoglycoprotein receptors, and thereby accumulate in the serum [95]. The most important function of sialic acid is probably its ability to act as a biological mask, via steric hindrance and electrostatic repulsion. One may also speculate that such changes make the Ig pathogenic by influencing their conformation.

In addition, revision of the Ig genes coding for the BCR rescues autoreactive B cells in the periphery, aided by the recombination-activating genes (RAG)1- and RAG2encoded enzymes [96]. Triple-nested reverse-transcriptase polymerase-chain reactions in single-sorted lymphocytes eluted from the salivary glands of SjS patients detected mRNAs for RAG1 and RAG2 in single B cells, but not in neighboring T cells [97], and their protein products were also identified in the salivary glands. Thus, ongoing B-cell stimulation may overwhelm the capacity of revision to prevent autoimmunity.

5.3.3 Intrinsic Abnormalities of B Cells in Primary SjS

The last three arguments against B cells as the main lymphocyte population in SjS are abnormalities detected in their molecular mechanisms. Pre-switch Ig transcripts are aberrantly retained in circulating post-switch memory B lymphocytes, although an ongoing switch is unlikely in the absence of transcripts for Bcl-6 [98] and AID [21]. Coexistence of transcripts for the two isotypes may perhaps be ascribed to a B cell intrinsic defect.

B cells are also peculiar in that their BCR partition, along with other proteins involved in cell signaling, partition into cholesterol and ganglioside M1-enriched microdomains of the cell membrane [99], termed lipid rafts (LRs). Interestingly, the association of the BCRs with the LRs persists, prolonging the signal transduction [33]. Finally, the single-cell analysis technology has proved that mRNA for BAFF exists in B cells from salivary glands. For this to occur, the downstream negative regulator of BAFF-mediated B-cell survival has to be inhibited [100].

Equally important in distinguishing the role of B cells in the pathogenesis of SjS is that BAFF potentiates the B cell selection with the BCR complex [101], and synergizes with IL-21 at that site [102]. Growing evidence suggests that the signaling mechanisms that maintain B-cell fitness during selection at transitional stages, and survival after maturation rely on cross-talk between BCR and one of the receptors for BAFF (recently highlighted in [103]). Of important note in this regard, there is an inverse correlation between the preexisting levels of BAFF and the number of months B cells take to repopulate the blood of rituximab-treated patients with primary SjS [104]. Supporting this concept are that some patients resist rituximab due to the local overexpression of BAFF in their salivary glands [105], and that human CD20-Tg autoimmune-prone mice with elevated levels of BAFF are resistant to rituximab, a monoclonal antibody directed against CD20 [106]. Thus, B cell infiltration would be modulated by BAFF in salivary glands [107], but not in lupus nephritis [108].