Viral and bacterial pathogens cause inflammation via Toll-like receptor (TLR) signaling. We have shown that effective responses to LPS may depend on cooperative interactions between TLR-expressing leukocytes and TLR-negative tissue cells. The aim of this work was to determine the roles of such networks in response to agonists of TLRs associated with antiviral and autoimmune responses. The TLR3 agonist poly(I:C) activated epithelial cells, primary endothelial cells, and two types of primary human smooth muscle cells (airway [ASMC] and vascular) directly, while the TLR7/8 agonist R848 required the presence of leukocytes to activate ASMC. In keeping with these data, ASMC expressed TLR3 but not TLR7 or TLR8. Activation of ASMC by poly(I:C) induced a specific cytokine repertoire characterized by induction of CXCL10 generation and the potential to recruit mast cells. We subsequently explored the ability of TLR agonists to cooperate in the induction of inflammation. Dual stimulation with LPS and poly(I:C) caused enhanced cytokine generation from epithelial and smooth muscle cells when in the presence of leukocytes. Thus, inflammatory responses to pathogens are regulated by networks in which patterns of TLR expression and colocalization of tissue cells and leukocytes are critical.