Palpable swelling of regional lymph nodes is a common sequela of microbial infections but the mechanism responsible for the sequestration and subsequent coordination of lymphocyte responses within these dynamic structures remains poorly understood. Here we show that draining lymph nodes of mast cell-deficient mice did not demonstrate swelling after intradermal bacterial challenge. Testing of individual mast cell-derived products in this model indicated that tumor necrosis factor was the main mediator of nodal hypertrophy, whereas tryptase and histamine had no effect. After peripheral mast cell activation, both tumor necrosis factor concentrations and the recruitment of circulating T cells were increased within draining nodes. These results show a critical function for peripheral mast cell-derived tumor necrosis factor in regulating the hypertrophy of draining lymph nodes during infection.

Mast cells (MCs) have recently received recognition as prominent effectors in the regulation of immune cell migration to draining lymph nodes and lymphocyte activation. However, their role in the development of humoral immune responses is not clear. Here, we demonstrate that subcutaneous or nasal administration of small-molecule MC activators with vaccine antigens evokes large increases in antigen-specific serum immunoglobulin G (IgG) responses. These responses were MC dependent and correlated with increased dendritic cell and lymphocyte recruitment to draining lymph nodes. Nasal instillation of these formulations also evoked antigen-specific secretory IgA and provided protection against anthrax lethal toxin challenge in vitro and against vaccinia virus infection in vivo. Collectively, these results define the MC as an integral sensory arm of the adaptive immune system. Moreover, they highlight MC activators as a new class of vaccine adjuvants, capable of inducing protective antigen-specific immune responses through needle-free routes of administration.

gp96 (GRP94) elicits antigen-presenting cell (APC) activation and can direct peptides into the cross- presentation pathways of APC. These responses arise through interactions of gp96 with Toll-like (APC activation) and endocytic (cross-presentation) receptors of APC. Previously, CD91, the alpha2-macroglobulin receptor, was identified as the heat shock/chaperone protein receptor of APC. Recent data indicates, however, that inhibition of CD91 ligand binding does not alter gp96 recognition and uptake. Furthermore, CD91 expression is not itself sufficient for gp96 binding and internalization. We now report that scavenger receptor class-A (SR-A), a prominent scavenger receptor of macrophages and dendritic cells, serves a primary role in gp96 and calreticulin recognition and internalization. gp96 internalization and peptide re-presentation are inhibited by the SR-A inhibitory ligand fucoidin, although fucoidin was without effect on alpha2-macroglobulin binding or uptake. Ectopic expression of SR-A in HEK 293 cells yielded gp96 recognition and uptake activity. In addition, macrophages derived from SR-A-/- mice were substantially impaired in gp96 binding and uptake. These data identify new roles for SR-A in the regulation of cellular responses to heat shock proteins.

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds to many proteins, some of which trigger signal transduction. Receptor-recognized forms of ␣ 2 -Macroglobulin (␣ 2 M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni 2؉ nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block ␣ 2 M*-induced signal transduction. In the current study, we employed ␣ 2 -macroglobulin (␣ 2 M)-agarose column chromatography to purify cell surface membrane binding proteins from 1-LN human prostate cancer cells and murine macrophages. The predominant binding protein purified from 1-LN prostate cancer cells was Grp 78 with small amounts of LRP, a fact that is consistent with our previous observations that there is little LRP present on the surface of these cells. The ratio of LRP:Grp 78 is much higher in macrophages. Flow cytometry was employed to demonstrate the presence of Grp 78 on the cell surface of 1-LN cells. Purified Grp 78 binds to ␣ 2 M* with high affinity (K d ϳ150 pM). A monoclonal antibody directed against Grp 78 both abolished ␣ 2 M*-induced signal transduction and co-precipitated LRP. Ligand blotting with ␣ 2 M* showed binding to both Grp 78 and LRP heavy chains in these preparations. Use of RNA interference to silence LRP expression had no effect on ␣ 2 M*-mediated signaling. We conclude that Grp 78 is essential for ␣ 2 M*-induced signal transduction and that a "co-receptor" relationship exists with LRP like that seen with several other ligands and receptors such as the uPA/uPAR (urinary type plasminogen activator or urokinase/uPA receptor) system.1 is a plasma proteinase inhibitor with broad specificity. Upon binding to proteinases, it undergoes a major conformational change that exposes receptor recognition sites on the molecule (1). This activated form of ␣ 2 M is designated ␣ 2 M*. Though difficult to reconcile, accumulating evidence has demonstrated that ␣ 2 M*-induced signaling occurs via a receptor that is functionally unique from the previously characterized ␣ 2 M* receptor, the low density lipoprotein receptor-related protein (LRP). LRP is a scavenger receptor (1) that binds to a variety of proteins, many of which trigger signal transduction (2-5). This pathway requires the activation of a pertussis toxin-sensitive G protein (2-5). The receptor-associated protein (RAP) that blocks the binding of all known ligands to LRP is also an antagonist for this signaling pathway (2-3). Whereas ␣ 2 M* binds to LRP, when cells are exposed to ␣ 2 M* a distinct set of signaling events is observed that differ from those induced by other ligands for this receptor (2-6). These include activation of a different G protein and the lack of antagonism by RAP or Ni 2ϩ (2-7). In addition, binding studies with a variety of cells in culture demonstrate two classes of binding sites, one of very high affinity (K d ϳ100 pM and 1600 sites/cell) and one of lower affinity (K d ...

GRP94(gp96) elicits CD8+ T cell responses against its bound peptides, a process requiring access of its associated peptides into the MHC class I cross-presentation pathway of APCs. Entry into this pathway requires receptor-mediated endocytosis, and CD91 (low-density lipoprotein receptor-related protein) has been reported to be the receptor mediating GRP94 uptake into APC. However, a direct role for CD91 in chaperone-based peptide Ag re-presentation has not been demonstrated. We investigated the contribution of CD91 to GRP94 cell surface binding, internalization, and trafficking in APCs. Whereas a clear role for CD91 in α2-macroglobulin binding and uptake was readily obtained, the addition of excess CD91 ligand, activated α2-macroglobulin, or receptor-associated protein, an antagonist of all known CD91 ligands, did not affect GRP94 cell surface binding, receptor-mediated endocytosis, or peptide re-presentation. These data identify a CD91-independent, GRP94 internalization pathway that functions in peptide Ag re-presentation.

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