Neutrophils play a crucial early role during the innate response, but little is known about their possible contribution when an adaptive immune response is installed. A robust neutrophilia and a T helper 1 (Th1) immune response are present after immunization with Complete Freund Adjuvant (CFA). We show that when FITC-labeled OVA was injected into the footpad of OVA/CFA immunized mice, the main OVA-FITC+ cells recruited in draining popliteal lymph nodes (LNs) were neutrophils, with most of them arriving at the LN by means of lymphatic vessels. The development of this OVA-FITC+ neutrophil influx requires an immune response against OVA. The OVA-FITC+ neutrophils present in LNs displayed mainly intracellular TNF-alpha, and their depletion resulted in an increase in the specific IL-5 levels. These data provide new evidence about the role played by neutrophils in vivo in adaptive immunity.

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

Synopsis The parasite Giardia lamblia possesses peripheral vacuoles (PVs) that function as both endosomes and lysosomes and are implicated in the adaptation, differentiation, and survival of the parasite in different environments. The mechanisms by which Giardia traffics essential proteins to these organelles and regulates their secretion have important implications in the control of parasite dissemination. In this study, we describe the participation of the heterotetrameric clathrin-adaptor protein gAP2 complex in lysosomal protein trafficking. A specific monoclonal antibody against the medium subunit (gμ2) of gAP2 showed localization of this complex to the PVs, cytoplasm, and plasma membrane in the growing trophozoites. gAP2 also colocalized with clathrin in the PVs, suggesting its involvement in endocytosis. Uptake experiments using standard molecules for the study of endocytosis revealed that gAP2 specifically participated in the endocytosis of LDL. Targeted downregulation of the gene encoding gμ2 in growing and encysting trophozoites resulted in a large decrease in the amount of cell growth and cyst wall formation, suggesting a distinct mechanism in which gAP2 is directly involved in both endocytosis and vesicular trafficking.