Summary We identify the interfollicular (IF) zone as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first two days post-immunization, antigen-specific T and B cells remained confined within the IF zone, formed long-lived interactions, and upregulated the transcriptional repressor Bcl6. T cells also acquired the Tfh cell markers CXCR5, PD-1 and GL7. Responding B and T cells migrated to the follicle interior directly from the IF zone, T cell immigration preceding B cells by one day. Notably, in the absence of cognate B cells, Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS and GL7 were no longer expressed on follicular Bcl6hi T cells that nevertheless persisted in the follicle. Thus, Ag-specific B cells are required for the maintenance of the PD-1hi ICOShi GL7hi Tfh cell phenotype within the follicle, but not for their initial differentiation in the IF zone.

Profound thrombocytopenia occurs in humans with sepsis and in mice administered lipopolysaccharide (LPS). Growing evidence indicates that platelets may contribute to these abnormalities, but whether that is a direct result of LPS activation of platelets or an indirect result of other inflammatory mechanisms remains unclear. Here we demonstrate that although platelets do not increase P-selectin expression in response to LPS, platelets bind more avidly to fibrinogen under flow conditions in a Toll-like receptor-4 (TLR4)-dependent manner. In addition, we find that CD41 ؉ megakaryocytes grown from fetal livers and adult circulating platelets express significant amounts of TLR4. LPS induced thrombocytopenia in wild-type mice but not in TLR4-deficient (TLR4 def ) mice. Wild-type platelets accumulated in the lungs of wild-type mice in response to LPS; TLR4 def platelets did not. However, wild-type platelets did not accumulate in the lungs of LPS-treated TLR4 def mice. Neutrophils also accumulated in the lungs, and this preceded platelet accumulation. Neutrophil depletion completely abolished LPS-induced platelet sequestration into the lungs, but platelet depletion did not affect neutrophil accumulation. Thus, our data show for the first time that platelets do express functional levels of TLR4, which contribute to thrombocytopenia through neutrophil-dependent pulmonary sequestration in response to LPS. ( IntroductionLipopolysaccharide (LPS) or endotoxin is the main structural component of Gram-negative bacteria and an important player in the ability of host cell detection of these foreign pathogens. LPS may also play a fundamental role in sepsis. LPS recognition by mammalian cells occurs through a multiprotein interaction. First, a plasma protein, LPS-binding protein (LBP), binds LPS and transfers LPS monomers to CD14. 1 CD14 is a high-affinity receptor for LPS present both as a soluble form in blood or as a glycophosphoinositol (GPI)-anchored protein on the surfaces of myeloid lineage cells. Indeed, CD14 Ϫ/Ϫ mice are at least 100 times more resistant to LPS-induced death. 2 However, LPS signaling in cells can only occur if the transmembrane molecule TLR4 is activated. TLR4 belongs to the family of Toll-like receptors that are type 1 transmembrane proteins characterized by an extracellular domain containing multiple leucine-rich repeats, a single transmembrane domain, and an intracellular Toll/interleukin-1 (IL-1) receptor (TIR) domain. Stimulation of TLR4 by LPS activates a signaling cascade that is characterized by the production of proinflammatory cytokines and subsequent immune response. The importance of TLR4 in LPS-induced signaling is emphasized by the fact that TLR4 def mice (C57BL/10ScCr), TLR4 knockout mice, and mice with a single point mutation in the TLR4 gene (C3H/HeJ) are resistant to the immunostimulatory and pathophysiologic effects of LPS. [3][4][5] TLR4 is present in many different cell types, including dendritic cells, neutrophils, macrophages, epithelial cells, keratinocytes, and endothelial cells. ...

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44−/− and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44−/− mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44−/− mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44−/− mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44−/− mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.

Experimental autoimmune encephalomyelitis (EAE) is mediated by inflammatory cells recruited from the circulation to the CNS. We used intravital microscopy to investigate the mechanisms of this recruitment. No leukocyte rolling and very little adhesion was observed in healthy control mice. In contrast, both rolling and adhesion was observed in brain postcapillary venules before onset of physical symptoms of EAE. Rolling and adhesion remained elevated for 2 wk and returned to near normal levels by 5 wk postsymptom onset. Consistent with a role for P-selectin in recruitment to the CNS, P-selectin protein was detected in the brains and spinal cords of EAE mice. Expression was highest before symptom onset and decreased over the next 2 wk. The importance of α4 integrin increased with time as anti-α4 integrin blocked ∼20, 50, and 60% of leukocyte rolling 2 days before disease onset, 5 days and 2 wk postonset of symptoms, respectively, and 85% of rolling 5 wk postsymptoms. Addition of anti-P-selectin to α4 integrin Ab-treated mice blocked all remaining rolling at each time point. Interestingly, however, α4 integrin-mediated rolling appeared to be entirely dependent on P-selectin as anti-P-selectin alone was able to completely block all leukocyte rolling. In the absence of rolling (with P-selectin Ab), a 70% reduction in adhesion was noted. A very similar reduction was seen when mice were treated with α4 integrin-blocking Ab. In conclusion, we describe increased leukocyte trafficking in the brains of EAE mice with important overlapping roles for both P-selectin and α4 integrin in mediating leukocyte-endothelial cell interactions.