Mechanisms and functions of lymphangiogenesis in regulating the immune response and inflammation resolution

Abstract

Research on inflammation-induced lymphangiogenesis in lymph nodes (LNs) has been largely centered on the early phases of inflammation, subcapsular lymphatics remodeling on the afferent side and its attendant effects on the immune response. Consequently, how cortical and medullary sinuses, which serve as exits for lymphocytes, are remodeled during inflammation and how this impacts the immune response and inflammation resolution remain unexplored questions. When we induced inflammation by footpad immunization, expansion of lymphatics within draining LNs persisted beyond 90 days. Analysis of the lymphatic vessel network within inflamed LNs revealed that the subcapsular sinuses and medullary and cortical sinuses were differentially expanded during the early and late phases of inflammation, respectively. Expansion of the subcapsular sinuses during early inflammation augmented dendritic cell migration into inflamed LNs. During later and prolonged inflammation, the more predominant expansion of medullary and cortical sinuses supported lymphocyte egress, restoring it to steady state levels following an initial phase of retention. In addition, such restoration of lymphocyte egress from inflamed LNs was demonstrated to be similar for both antigen-activated and naive T cells. Preferential expansion of cortical and medullary sinuses during prolonged inflammation was a dynamic process dependent on crosstalk and synergy between fibroblastic reticular cells (FRCs), interstitial flow and vascular endothelial growth factor-A (VEGF-A). Our data sheds new light on the biological significance of LN lymphangiogenesis during prolonged inflammation and further underscores the collaborative roles of lymphatics and FRCs in modulating LN plasticity and function. In an earlier study, we showed that B cells were critical for initiating lymphangiogenesis within the draining LN during early inflammation induced by footpad immunization. Here we discovered that during later phases of inflammation, neutrophils accumulate in inflamed B cell deficient (?MT) LNs and can substitute for B cells in driving lymphangiogenesis. Neutrophils do not migrate to wild type LNs during inflammation and hence did not play a role in mediating LN lymphangiogenesis in these mice. However neutrophils accumulate in immunized WT footpads and in sensitized skin and were found in these primary sites of inflammation, to be critical for organizing lymphangiogenesis and consequently, inflammation resolution. We noted that the absence of neutrophils did not seem to alter the amount of VEGF-A and VEGF-C in the inflamed footpad. Instead, the absence of neutrophils resulted in an obvious reduction in MMP-9, a proteolytic enzyme involved in extracellular matrix remodeling. Subsequent experiments revealed that neutrophils were the principal source of MMP-9 in the inflamed footpads. Furthermore, such neutrophil-derived MMP-9 was likely to be constitutively active as it was not associated with TIMP-1, an inhibitor of MMP activity. In vitro experiments further revealed the unexpected finding that neutrophils are able to produce, in addition to MMP-9, VEGF-C upon activation.