Trafficking, Not Lymphoproliferation, Promotes Lymphadenopathy in Idiopathic Multicentric Castleman Disease

Idiopathic multicentric Castleman disease (iMCD) is a rare and life-threatening hematologic illness characterized by multifocal lymphadenopathy for an unknown cause. Patients with iMCD experience periods of systemic inflammation due to cytokine release that includes interleukin-6 (IL-6). Treatment with IL-6 inhibition is effective in only one-half of patients and thus, a more complete understanding of the disease process of iMCD is urgently needed to advance the development of treatment options for refractory patients.

Though not well understood, iMCD is often described as a lymphoproliferative disorder and assumed to be due to expansion of lymphocytes in lymph nodes. To determine what contributes to the characteristic lymphadenopathy in iMCD, we investigated factors that influence lymph node size including cell proliferation and chemotaxis.

To assess whether lymphadenopathy in iMCD was associated with cell proliferation, we analyzed lymph node size and fluorodeoxyglucose (FDG) uptake from our CD patient natural history registry and published case reports. Analysis of 273 patients (iMCD, n=133; unicentric CD, n=70; lymphoma, n=16; other inflammatory diseases, n=54), revealed that there were no significant differences in maximum lymph node size between groups. Comparison of the FDG maximum standardized uptake value indicated that lymph nodes from iMCD patients had 2.8-fold lower mean FDG avidity than lymphoma. We also measured cell proliferation directly by performing immunohistochemistry with Ki-67 on iMCD (n=15), lymphoma (n=9), and benign, sentinel (n=10) lymph nodes. We identified Ki-67 positive and negative nuclei by developing an algorithm to automatedly segment nuclei and measure Ki-67 levels. We discovered that the frequency of Ki-67 positive cells in iMCD lymph nodes (4.1%) was similar to the negative control sentinels (3.8%), yet significantly lower compared to lymphoma (31.8%). These data indicate that although lymph nodes from lymphoma and iMCD patients were similar in size, mechanisms other than local cell expansion, such as increased cellular mobilization, may contribute to lymphadenopathy in iMCD.

We previously showed that 3 of the top 20 cytokines or chemokines found in iMCD directly promote chemotaxis (CXCL13, CCL19 and CCL21) suggesting that increased trafficking to the tissue may influence lymph node size in iMCD. As CXCL13, CCL19, and CCL21 function to attract B and T cells to specific regions of secondary lymphoid organs, we profiled gene expression in discrete regions of the lymph node tissue where these chemokines are known to be expressed using Nanostring GeoMx technology. Analysis of tissue from iMCD patients (n=6) versus reactive, non-specific inflammatory controls (n=3) revealed increased gene expression of CXCL13, CCL19, and CCL21 in the germinal center. Furthermore, CXCL13 expression was elevated in the interfollicular space reaching similar levels as control germinal centers. As CXCL13 is typically expressed in the germinal center whereas CCL19 and CCL21 are most notably confined to the interfollicular space, our data suggest that the expression of these factors are dysregulated in overall levels and pattern within the tissue, which may result in attracting lymphocytes out of circulation and into the lymph node.

To determine if there were any changes in cell populations that respond to these chemoattractants, we profiled chemokine receptor expression on circulating immune cell types in iMCD (n=13) and healthy controls (n=17) by flow cytometry. Expression of CXCR5, the cognate receptor to CXCL13, was significantly reduced among circulating B and T cells in iMCD during disease flare and restored in remission. Other chemokine receptors were also decreased, including CCR7, which responds to CCL19 and CCL21 ligands, CXCR3 and CCR6 in lymphocyte subsets. These data may indicate that there is an increase in lymphocyte trafficking to lymph node tissue or other regions of heightened chemokine expression.

Together, our data suggest that lymphocyte mobilization, rather than lymphoproliferation, contributes to lymphadenopathy in iMCD. Dysregulated lymphocyte trafficking and disorganization in secondary lymphoid organs may contribute to flare episodes and iMCD pathogenesis. As such, targeting the CXCR5/CXCL13 axis is a potentially interesting therapeutic strategy for the treatment of iMCD.