Inflammatory cytokines IL-6 and TNF-α regulate lymphocyte trafficking through the local lymph node

Abstract

Lymphocyte trafficking from blood to lymph and back is a tightly regulated process. Given appropriate stimuli, trafficking of cells through the lymph node changes from a ‘steady-state’ to a bimodal flow. Initially, a ‘shutdown’ phase occurs, leading to a dramatic reduction in efferent cell output. This is followed by a ‘recruitment’ phase whereby the efferent cell output becomes greatly elevated before returning to baseline levels. The shutdown/recruitment process is hypothesised to promote encounters between Ag-specific lymphocytes and APCs in an environment conducive to immune response induction. Cytokines, such as TNF-α have been shown to play an important role in regulating lymphocyte trafficking. Here, we unravel the role of cytokines in the regulation of cell trafficking using an in vivo sheep lymphatic cannulation model whereby the prefemoral lymph nodes were cannulated and recombinant cytokines were injected subcutaneously into the draining area of the cannulated node. We demonstrate that local injection of purified IL-6 or TNF-α stimulates shutdown/recruitment in the draining lymph node. While the effect of IL-6 appears to be direct, TNF-α may mediate shutdown/recruitment through IL-6.

Introduction

It is widely accepted that immune induction is initiated in the draining lymph node, often the first lymphoid organ encountered by a pathogen. Within the compact body of the node, activated APCs including dendritic cells (DCs) engage lymphocytes in a complex interplay to generate effector cells that provide appropriate and specific responses against the infection (reviewed in (Lammermann and Sixt, 2008, Millington et al., 2007, von Andrian and Mempel, 2003)). When a pathogen or foreign Ag gains entry into the body, for example via epithelial surfaces such as the skin, it is captured and processed by DCs (Banchereau and Steinman, 1998). Guided by inflammatory signals, DCs migrate to the draining lymph node and display the Ag and additional stimulatory molecules on their cell surface. Lymphocytes, for their part, cross the post-capillary venules (Girard and Springer, 1995) and traverse the node in search of DCs presenting cognate Ag and co-stimulatory signals. In the absence of Ag-specific stimuli, lymphocytes leave the node undifferentiated and continue recirculating. In contrast, the presence of appropriate signals leads to the activation and proliferation of Ag-specific lymphocytes. These lymphocytes then leave the node and home to tissues where effector responses are required (Bujdoso et al., 1989, Campbell and Butcher, 2002).

The lymphatic cannulation model in sheep allows the study of lymphocyte trafficking through the node in ‘real-time’ under stimulatory conditions, by collecting lymph as it leaves the lymph node. One notable change in lymphocyte trafficking that occurs in the presence of appropriate stimuli is a dramatic switch from a ‘steady-state’ flow (random fluctuation about a basal value) to a bi-modal flow (Bujdoso et al., 1989, Cahill et al., 1976, Mackay et al., 1992). The initial ‘shutdown’ phase, characterised by severe reduction in cell output from lymph node, occurs despite increased recruiting of lymphocytes from the blood into the lymph node (Cahill et al., 1976, Mackay et al., 1992). The shutdown phase is followed by a recruitment phase, during which the cell output of the lymph node is greatly increased. The shutdown phase is rapidly induced but is usually transient while the recruitment phase may last for a period of hours to days following stimulation (Windon et al., 2001). While shutdown/recruitment were initially thought to be mediated through Ag, it was shown that this process could also be driven by adjuvants alone (Windon et al., 2000).

Real-time imaging of cell movements within the lymphoid body have highlighted the importance of the micro-anatomy of the lymph node, charted the spatial and temporal dynamics of immune cell to cell contact within the node, and defined the adhesion cascades that promote cell trafficking and contact (see reviews (Lammermann and Sixt, 2008, Mempel et al., 2004, Millington et al., 2007, von Andrian and Mempel, 2003)). However, the contribution of cytokines to the regulation of the cell activities in lymph node in real-time, remains an area less explored to date (Randolph et al., 2005).

IL-6 and TNF-α are two cytokines traditionally considered key mediators of acute inflammation. Both factors are also crucial to the development of specific immune responses, as mice lacking IL-6 or TNF-α gene expression are deficient in T and B cell function (Kopf et al., 1995, Pasparakis et al., 1996). Inflammation and immune induction can be viewed as intimately linked events as the successful resolution of the inflammatory process leads to the transition from innate to adaptive immunity (Hoebe et al., 2004). Of the two cytokines, IL-6 in particular, through differential control of leukocyte recruitment, activation and apoptosis, has emerged as one of a network of mediators directing this shift from innate to specific immunity (Jones, 2005). Importantly, IL-6 has also been implicated in the regulation of lymphocyte trafficking through the lymph node following an inflammatory stimulus (Windon et al., 2000, Windon et al., 2001). In one study where the adjuvant ISCOMATRIX™ and influenza Ag (H3N2 strain) were injected into the area draining the prefemoral lymph node, a prolonged shutdown for 24–48 h followed by a period of increased cell output was observed (Windon et al., 2001) and the initiation of shutdown was found to be closely associated with IL-6 expression.

Given that the activities of IL-6 and TNF-α cytokines are closely linked (Brouckaert et al., 1993, Moller and Villiger, 2006) and can also influence the expression levels of one another, we hoped to dissect the roles of IL-6 and TNF-α in lymphocyte trafficking through the local lymph node by employing purified recombinant forms of the cytokines in a sheep lymphatic cannulation model. Our results indicate that both these pro-inflammatory cytokines influence lymph node shutdown; however, while IL-6 appears to act directly to induce shutdown, TNF-α may function through IL-6.