In host defence, pattern recognition plays an essential role by enabling the immune system to discriminate self from pathogenic non-self. Pattern recognition is mediated by leukocyte expressed pattern recognition molecules (PRMs), which recognise pathogen associated molecular patterns (PAMPs) on pathogens. Phagocytosis is a critical event for anti-microbial defence and its contribution is not limited to the clearance and killing of pathogens, but extends to the activation of adaptive immunity through production of pro-inflammatory mediators and antigen presentation. Anti- fungal immunity is extremely efficient and operates via recognition, phagocytosis and killing of fungal pathogens by leukocytes. We have examined Dectin-1, a non-opsonic pattern recognition receptor that recognises live fungi and fungal derived particles and that is highly expressed on various leukocyte populations. We wanted to establish whether Dectin-1 contributes to anti-fungal defence by analysing various aspects of the receptor biology. Using both confocal microscopy and flow-cytometry, we demonstrate that Dectin-1 is a phagocytic receptor. Furthermore, using cell lines expressing receptor mutants, we show that this capacity is mediated by the membrane proximal tyrosine residue located in the ITAM-like motif. This makes Dectin-1 the first described phagocytic leukocyte expressed receptor for unopsonised fungi and fungal derived particles, and the first pattern recognition receptor that mediates phagocytic uptake through a tyrosine based motif. We demonstrate that the mechanisms by which Dectin-1 mediates cytoskeletal activation and actin polymerisation are novel, and not shared with the canonical IT AM containing Fc(gamma)Rs. In particular the observation that Syk kinase plays not role in Dectin- 1 mediated phagocytosis in macrophages. We show that Dectin-1 mediates cellular activation in response to zymosan particles and that these (beta)-glucan dependent biological effects require collaboration with toll-like receptors (TLRs) at the cell surface. We also show that ligand size determines intracellular receptor trafficking following internalisation. Furthermore, we show that when biologically active soluble glucans are internalised by Dectin-1, the receptor is retained intracellularly yet, when biologically silent glucans are used, Dectin-1 is recycled. Dectin-1 is thus established as both an important phagocytic fungal pattern recognition receptor with pro- inflammatory abilities and an additional tool with which to study the diversity of signalling processes associated with leukocyte expressed receptors.

Regulated adherence of monocytes to extracellular matrix macromolecules is a prerequisite for their accumulation at sites of pulmonary infection and inflammation. To begin to assess the pathobiological importance of alterations in monocyte adherence to extracellular matrix in inflammatory lung diseases, the adherence properties of monocytes from patients with an inflammatory lung disease (bronchiectasis) and healthy subjects to a representative matrix component (fibronectin) were compared. Spontaneous adherence of monocytes from the control subjects was 20 to 25%, whereas that of the patients' cells was 2 to 3-fold higher and correlated with the severity of airway inflammation. Endotoxin (LPS) and cytokines from areas of airway disease are likely to be responsible for the observed monocyte activation since: 1) LPS was detected in plasma from all of the patients but none of the control subjects; and 2) LPS and cytokines produced dose-related increases in the adherence of normal monocytes in vitro. Monocyte adherence to fibronectin was substantially mediated by CD11/CD18 integrins, via both RGD-dependent and RGD-independent mechanisms. These data indicate that signals arising from foci of pulmonary inflammation are likely determinants of the accumulation of monocytes in the lungs of patients with chronic inflammatory lung diseases. There was a striking relationship between the adherence properties of monocytes and functions that are of biological importance at sites of inflammation. Spontaneously adherent monocytes had an "inflammatory effector" phenotype, non-adherent cells had an "immune modulatory" phenotype and monocytes that could stimulated to adhere by LPS (LPS-adherent cells) had an intermediate phenotype. In addition, only the adherent monocyte subpopulations were replete with HLE and these cells contained a substantial (10 to 11-fold) molar excess of HLE compared with the physiological inhibitor of this enzyme (a1-antitrypsin). Maturation in vitro increased the accumulation of a1-antitrypsin by all of the monocyte subpopulations. In contrast, proinflammatory mediators up-regulated a1-antitrypsin accumulation by only the spontaneously adherent cells, probably by translational or post-translational mechanisms. In conclusion, these data indicate that monocytes are heterogeneous in their ability to accumulate at sites of infection and inflammation. In addition, the capacity of monocytes to adhere to fibronectin is related to monocyte functions that are of biological importance at sites of infection and inflammation. Furthermore, LPS released from foci of infection, may induce the accumulation of monocytes with an inflammatory effector phenotype, and may thereby promote resolution of tissue infection. Alternatively, LPS may promote the recruitment of monocytes with capacity to contribute to HLE-mediated tissue injury.