Secondary lymphoid organ chemokine (SLC) is expressed in high endothelial venules and in T cell zones of spleen and lymph nodes (LNs) and strongly attracts naive T cells. In mice homozygous for the paucity of lymph node T cell (plt) mutation, naive T cells fail to home to LNs or the lymphoid regions of spleen. Here we demonstrate that expression of SLC is undetectable in plt mice. In addition to the defect in T cell homing, we demonstrate that dendritic cells (DCs) fail to accumulate in spleen and LN T cell zones of plt mice. DC migration to LNs after contact sensitization is also substantially reduced. The physiologic significance of these abnormalities in plt mice is indicated by a markedly increased sensitivity to infection with murine hepatitis virus. The plt mutation maps to the SLC locus; however, the sequence of SLC introns and exons in plt mice is normal. These findings suggest that the abnormalities in plt mice are due to a genetic defect in the expression of SLC and that SLC mediates the entry of naive T cells and antigen-stimulated DCs into the T cell zones of secondary lymphoid organs.

Human plasmacytoid dendritic cells (pDCs) are major producers of IFNα, are activated by CpG motifs, and are believed to enter lymph nodes (LNs) via L-selectin dependent extravasation across high endothelial venules. To identify a similar murine DC type, CD11c+ cells in the LNs of L-selectin–deficient and control BALB/c mice were compared, revealing a population of CD11c+CD11b− cells that is reduced 85% in the LNs of L-selectin–deficient mice. These cells are Gr-1+B220+CD19−, either CD4+ or CD8+, and localize within T cell zones of LNs. Freshly isolated CD11c+Gr-1+ cells express major histocompatibility complex class II at low levels, display a plasmacytoid morphology, and survive poorly in culture. Their survival is increased and they develop a DC-like morphology in interleukin 3 and CpG. Like human pDCs, CD11c+Gr-1+ cells stimulate T cell proliferation after activation with CpG and produce IFNα after stimulation with influenza virus. These cells also display a strain-specific variation in frequency, being fivefold increased in the LNs of BALB/c relative to C57BL/6 mice. These CD11c+CD11b−B220+Gr-1+ cells appear to be the murine equivalent of human pDCs.

Infection with pathogenic influenza virus induces severe pulmonary immune pathology, but the specific cell types that cause this have not been determined. We characterized inflammatory cell types in mice that overexpress MCP-1 (CCL2) in the lungs, then examined those cells during influenza infection of wild-type (WT) mice. Lungs of both naive surfactant protein C-MCP mice and influenza-infected WT mice contain increased numbers of CCR2+ monocytes, monocyte-derived DC (moDC), and exudate macrophages (exMACs). Adoptively transferred Gr-1+ monocytes give rise to both moDC and exMACs in influenza-infected lungs. MoDC, the most common inflammatory cell type in infected lungs, induce robust naive T cell proliferation and produce NO synthase 2 (NOS2), whereas exMACs produce high levels of TNF-α and NOS2 and stimulate the proliferation of memory T cells. Relative to WT mice, influenza-infected CCR2-deficient mice display marked reductions in the accumulation of monocyte-derived inflammatory cells, cells producing NOS2, the expression of costimulatory molecules, markers of lung injury, weight loss, and mortality. We conclude that CCR2+ monocyte-derived cells are the predominant cause of immune pathology during influenza infection and that such pathology is markedly abrogated in the absence of CCR2.

B lymphocytes re-circulate between B-cell-rich compartments (follicles or B zones) in secondary lymphoid organs, surveying for antigen. After antigen binding, B cells move to the boundary of B and T zones to interact with T-helper cells. Despite the importance of B--T-cell interactions for the induction of antibody responses, the mechanism causing B-cell movement to the T zone has not been defined. Here we show that antigen-engaged B cells have increased expression of CCR7, the receptor for the T-zone chemokines CCL19 and CCL21, and that they exhibit increased responsiveness to both chemoattractants. In mice lacking lymphoid CCL19 and CCL21 chemokines, or with B cells that lack CCR7, antigen engagement fails to cause movement to the T zone. Using retroviral-mediated gene transfer we demonstrate that increased expression of CCR7 is sufficient to direct B cells to the T zone. Reciprocally, overexpression of CXCR5, the receptor for the B-zone chemokine CXCL13, is sufficient to overcome antigen-induced B-cell movement to the T zone. These findings define the mechanism of B-cell relocalization in response to antigen, and establish that cell position in vivo can be determined by the balance of responsiveness to chemoattractants made in separate but adjacent zones.

Upon TCR-mediated positive selection, developing thymocytes relocate within the thymus from the cortex to the medulla for further differentiation and selection. However, it is unknown how this cortex–medulla migration of thymocytes is controlled and how it controls T cell development. Here we show that in mice deficient for CCR7 or its ligands mature single-positive thymocytes are arrested in the cortex and do not accumulate in the medulla. These mutant mice are defective in forming the medullary region of the thymus. Thymic export of T cells in these mice is compromised during the neonatal period but not in adulthood. Thymocytes in these mice show no defects in maturation, survival, and negative selection to ubiquitous antigens. TCR engagement of immature cortical thymocytes elevates the cell surface expression of CCR7. These results indicate that CCR7 signals are essential for the migration of positively selected thymocytes from the cortex to the medulla. CCR7-dependent cortex–medulla migration of thymocytes plays a crucial role in medulla formation and neonatal T cell export but is not essential for maturation, survival, negative selection, and adult export of thymocytes.

Rationale: In humans, immune responses to inhaled aeroallergens develop in the lung and draining lymph nodes. Many animal models of asthma bypass this route and instead use intraperitoneal injections of allergen using aluminum hydroxide as an adjuvant. Objectives: We investigated whether allergic sensitization through the airway elicits immune responses qualitatively different than those arising in the peritoneum. Methods: Mice were sensitized to allergen through the airway using low-dose LPS as an adjuvant, or through the peritoneum using aluminum hydroxide as an adjuvant. After a single allergen challenge, ELISA and flow cytometry were used to measure cytokines and leukocyte subsets. Invasive measurements of airway resistance were used to measure allergen-induced airway hyperreactivity (AHR). Measurements and Main Results: Sensitization through the peritoneum primed strong Th2 responses and eosinophilia, but not AHR, after a single allergen challenge. By contrast, allergic sensitization through the airway primed only modest Th2 responses, but strong Th17 responses. Th17 cells homed to the lung and released IL-17 into the airway on subsequent encounter with inhaled allergen. As a result, these mice developed IL-17-dependent airway neutrophilia and AHR. This AHR was neutrophil-dependent because it was abrogated in CXCR2-deficient mice and also in wild-type mice receiving a neutrophil-depleting antibody. Individually, neither IL-17 nor ongoing Th2 responses were sufficient to confer AHR, but together they acted synergistically to promote neutrophil recruitment, eosinophil recruitment and AHR. Conclusions: Allergic sensitization through the airway primes modest Th2 responses but strong Th17 responses that promote airway neutrophilia and acute AHR. These findings support a causal role for neutrophils in severe asthma. Keywords: asthma; lung; immunityThe most widely used mouse model of asthma involves allergic sensitization by intraperitoneal injections of ovalbumin (OVA) complexed with the Th2 adjuvant, aluminum hydroxide (alum) (8). These animals are typically challenged by intranasal instillation, or an aerosol, of OVA. Variations of this model have been used for many years and have been valuable for studying Th2-mediated responses in allergic pulmonary inflammation. However, some features of this model are inconsistent with human asthma. For example, the level of airway eosinophils can reach 80% in this mouse model (8), whereas in human patients it is rarely greater than 5% (9). Also, in this model, neutrophil recruitment to the airway is transient (10) and dispensable for airway hyperreactivity (AHR) (11). Therefore the alum-mediated model is not useful for studying the function of neutrophils in asthma. We hypothesized that some differences between this mouse model and human asthma might reflect the impact of the unique lung environment on developing immune responses. For example, alveolar macrophages and airway epithelial cells produce the regulatory cytokine, 13), which is required for the induction of both ...

T cell homing to peripheral lymph nodes (PLNs) is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial tethering and rolling via L-selectin, firm adhesion of T cells requires rapid upregulation of lymphocyte function–associated antigen 1 (LFA-1) adhesiveness by a previously unknown pathway that activates a Gαi-linked receptor. Here, we used intravital microscopy of murine PLNs to study the role of thymus-derived chemotactic agent (TCA)-4 (secondary lymphoid tissue chemokine, 6Ckine, Exodus-2) in homing of adoptively transferred T cells from T-GFP mice, a transgenic strain that expresses green fluorescent protein (GFP) selectively in naive T lymphocytes (TGFP cells). TCA-4 was constitutively presented on the luminal surface of HEVs, where it was required for LFA-1 activation on rolling TGFP cells. Desensitization of the TCA-4 receptor, CC chemokine receptor 7 (CCR7), blocked TGFP cell adherence in wild-type HEVs, whereas desensitization to stromal cell–derived factor (SDF)-1α (the ligand for CXC chemokine receptor 4 [CXCR4]) did not affect TGFP cell behavior. TCA-4 protein was not detected on the luminal surface of PLN HEVs in plt/plt mice, which have a congenital defect in T cell homing to PLNs. Accordingly, TGFP cells rolled but did not arrest in plt/plt HEVs. When TCA-4 was injected intracutaneously into plt/plt mice, the chemokine entered afferent lymph vessels and accumulated in draining PLNs. 2 h after intracutaneous injection, luminal presentation of TCA-4 was detectable in a subset of HEVs, and LFA-1–mediated TGFP cell adhesion was restored in these vessels. We conclude that TCA-4 is both required and sufficient for LFA-1 activation on rolling T cells in PLN HEVs. This study also highlights a hitherto undocumented role for chemokines contained in afferent lymph, which may modulate leukocyte recruitment in draining PLNs.

TH1-polarized immune responses, which confer protection against intracellular pathogens, are thought to be initiated by dendritic cells (DCs) that enter lymph nodes from peripheral tissues. We found following viral infection or immunization, inflammatory monocytes were recruited into lymph nodes directly from the blood to become CD11c+CD11bhiGr-1+ inflammatory DCs, which produced abundant interleukin 12(p70) and potently stimulated TH1 responses. This monocyte extravasation required CCR2 but not CCL2 or CCR7. Thus, inflammatory DC accumulation and TH1 responses were markedly reduced in Ccr2−/−mice, preserved in Ccl2−/− mice, and relatively increased in CCL19-CCL21-Ser-deficient plt mutant mice, in which all other lymph node DC types were reduced. We conclude that blood-derived inflammatory DCs play a major role in the development of TH1 immune responses.