A CXCL1 paracrine network links cancer chemoresistance and metastasis.

Metastasis and chemoresistance in cancer are linked phenomena, but the molecular basis for this link is unknown. We uncovered a network of paracrine signals between carcinoma, myeloid, and endothelial cells that drives both processes in breast cancer. Cancer cells that overexpress CXCL1 and 2 by transcriptional hyperactivation or 4q21 amplification are primed for survival in metastatic sites. CXCL1/2 attract CD11b(+)Gr1(+) myeloid cells into the tumor, which produce chemokines including S100A8/9 that enhance cancer cell survival. Although chemotherapeutic agents kill cancer cells, these treatments trigger a parallel stromal reaction leading to TNF-α production by endothelial and other stromal cells. TNF-α via NF-kB heightens the CXCL1/2 expression in cancer cells, thus amplifying the CXCL1/2-S100A8/9 loop and causing chemoresistance. CXCR2 blockers break this cycle, augmenting the efficacy of chemotherapy against breast tumors and particularly against metastasis. This network of endothelial-carcinoma-myeloid signaling interactions provides a mechanism linking chemoresistance and metastasis, with opportunities for intervention.

S100A8-S100A9 protein complex mediates psoriasis by regulating the expression of complement factor C3.

Psoriasis is a common heterogeneous inflammatory skin disease with a complex pathophysiology and limited treatment options. Here we performed proteomic analyses of human psoriatic epidermis and found S100A8-S100A9, also called calprotectin, as the most upregulated proteins, followed by the complement component C3. Both S100A8-S100A9 and C3 are specifically expressed in lesional psoriatic skin. S100A9 is shown here to function as a chromatin component modulating C3 expression in mouse and human cells by binding to a region upstream of the C3 start site. When S100A9 was genetically deleted in mouse models of skin inflammation, the psoriasis-like skin disease and inflammation were strongly attenuated, with a mild immune infiltrate and decreased amounts of C3. In addition, inhibition of C3 in the mouse model strongly reduced the inflammatory skin disease. Thus, S100A8-S100A9 can regulate C3 at the nuclear level and present potential new therapeutic targets for psoriasis.

ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia.

Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1-deficient mice were defective in these effector functions. Mechanistically, ICAM-1-mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Guanine nucleotide-binding proteins of the G12 family shape immune functions by controlling CD4+ T cell adhesiveness and motility.

Integrin-mediated adhesion plays a central role in T cell trafficking and activation. Genetic inactivation of the guanine nucleotide-binding (G) protein alpha-subunits Galpha(12) and Galpha(13) resulted in an increased activity of integrin leukocyte-function-antigen-1 in murine CD4(+) T cells. The interaction with allogeneic dendritic cells was enhanced, leading to an abnormal proliferative response in vitro. In vivo, T cell-specific inactivation of Galpha(12) and Galpha(13) caused lymphadenopathy due to increased lymph node entry and enhanced T cell proliferation, and the susceptibility toward T cell-mediated diseases was enhanced. Mechanistically, we show that in the absence of Galpha(12) and Galpha(13) the activity of the small GTPases Rac1 and Rap1 was increased, whereas signaling of the small GTPase RhoA was strongly reduced. Our data indicate that locally produced mediators signal through Galpha(12)- and Galpha(13)-coupled receptors to negatively regulate cell polarization and adhesiveness, thereby fine-tuning T cell trafficking, proliferation, and susceptibility toward T cell-mediated diseases.

A role for LFA-1 in delaying T-lymphocyte egress from lymph nodes.

Lymphocytes use the integrin leukocyte function-associated antigen-1 (LFA-1) to cross the vasculature into lymph nodes (LNs), but it has been uncertain whether their migration within LN is also LFA-1 dependent. We show that LFA-1 mediates prolonged LN residence as LFA-1(-/-) CD4 T cells have significantly decreased dwell times compared with LFA-1(+/+) T cells, a distinction lost in hosts lacking the major LFA-1 ligand ICAM-1. Intra-vital two-photon microscopy revealed that LFA-1(+/+) and LFA-1(-/-) T cells reacted differently when probing the ICAM-1-expressing lymphatic network. While LFA-1(+/+) T cells returned to the LN parenchyma with greater frequency, LFA-1(-/-) T cells egressed promptly. This difference in exit behaviour was a feature of egress through all assessed lymphatic exit sites. We show that use of LFA-1 as an adhesion receptor amplifies the number of T cells returning to the LN parenchyma that can lead to increased effectiveness of T-cell response to antigen. Thus, we identify a novel function for LFA-1 in guiding T cells at the critical point of LN egress when they either exit or return into the LN for further interactions.

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction.

Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation.

Chronic liver inflammation and hepatocellular carcinogenesis are independent of S100A9.

The S100A8/A9 heterodimer (calprotectin) acts as a danger signal when secreted into the extracellular space during inflammation and tissue damage. It promotes proinflammatory responses and drives tumor development in different models of inflammation-driven carcinogenesis. S100A8/A9 is strongly expressed in several human tumors, including hepatocellular carcinoma (HCC). Apart from this evidence, the role of calprotectin in hepatocyte transformation and tumor microenvironment is still unknown. The aim of this study was to define the function of S100A8/A9 in inflammation-driven HCC. Mice lacking S100a9 were crossed with the Mdr2(-/-) model, a prototype of inflammation-induced HCC formation. S100a9(-/-) Mdr2(-/-) (dKO) mice displayed no significant differences in tumor incidence or multiplicity compared to Mdr2(-/-) animals. Chronic liver inflammation, fibrosis and oval cell activation were not affected upon S100a9 deletion. Our data demonstrate that, although highly upregulated, calprotectin is dispensable in the onset and development of HCC, and in the maintenance of liver inflammation.

Chronic lymphocytic leukemia cells induce defective LFA-1-directed T-cell motility by altering Rho GTPase signaling that is reversible with lenalidomide.

T lymphocytes have an essential role in adaptive immunity and rely on the activation of integrin lymphocyte function-associated antigen-1 (LFA-1) to mediate cell arrest and migration. In cancer, malignant cells modify the immune microenvironment to block effective host antitumor responses. We show for the first time that CD4 and CD8 T cells from patients with chronic lymphocytic leukemia (CLL) exhibit globally impaired LFA-1-mediated migration and that this defect is mediated by direct tumor cell contact. We show that following the coculture of previously healthy T cells with CLL cells, subsequent LFA-1 engagement leads to altered Rho GTPase activation signaling by downregulating RhoA and Rac1, while upregulating Cdc42. Of clinical relevance, repair of this T-cell defect was demonstrated using the immunomodulatory drug lenalidomide, which completely rescued adhesion and motility function by restoring normal Rho GTPase activation signaling. Our report identifies a novel cancer immune evasion mechanism whereby tumor cells induce Rho GTPase signaling defects in T cells that prevent appropriate LFA-1 activation and motility. We believe these findings identify important biomarkers and highlight the clinical utility of immunotherapy to rescue normal T-cell function in CLLs that are likely to have relevance in other cancers.

Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation.

Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation. Conversely, in mice with clodronate-ablated macrophages, neutrophils extravasate, but have limited ability to reach the peritoneal fluid. Tissue macrophages synthesize neutrophil chemoattractants CXCL1/CXCL2 (CXC chemokine ligands 1/2) in response to LPS. Mast cells also produce these chemokines of which a proportion are preformed in granules. Release of the granules and new CXCL1/CXCL2 synthesis is Toll-like receptor 4-dependent. Both in vivo studies with blocking monoclonal antibodies and in vitro chemotaxis experiments show the neutrophil response to mast cells and macrophages to be CXCL1/CXCL2-dependent. The data are in keeping with the model that mast cells, optimally positioned in close proximity to the vasculature, initiate an early phase of neutrophil recruitment by releasing the chemoattractants CXCL1/CXCL2. Having arrived within the stimulated tissue, neutrophils penetrate further in a macrophage-dependent manner. Therefore, we demonstrate a positive role for mast cells in tissue inflammation and define how this comes about with contribution from a second tissue cell, the macrophage.

A new protective role for S100A9 in regulation of neutrophil recruitment during invasive pneumococcal pneumonia.

The S100A8/A9 heterodimer is abundantly expressed by myeloid cells, especially neutrophils, but its mechanism of action is only partially determined. In this study we investigated S100A8/A9 involvement in the host response to Streptococcus pneumoniae infection making use of S100a9(-/-) mice that lack heterodimer expression in myeloid cells. S100a9(-/-) mice that were infected intranasally with pneumococci rapidly succumbed, with 80% mortality after 48 h, whereas the majority of wild-type mice recovered. Over this time period, S100a9(-/-) mice displayed an average 6-fold reduction in circulating and lung-recruited neutrophils. Taqman analysis of S100a9(-/-) lungs revealed decreased production of a dominant subset of 5 cytokines and chemokines associated with neutrophil recruitment. The greatest differential was with the cytokine granulocyte colony-stimulating factor (G-CSF) that causes bone marrow release of neutrophils into the circulation (1900-fold difference at 48 h). Treating S100a9(-/-) mice with G-CSF reversed their increased susceptibility to infection by enhancing both circulating neutrophils and neutrophil recruitment into infected lungs, by reducing pneumococcal colony forming units, and by elevation of chemokine CXCL1, cytokine IL-6, and endogenous G-CSF proteins. Thus S100A9, potentially with its partner S100A8, makes a major contribution in the host response to pneumococcal infection by increasing circulating neutrophils principally regulation of G-CSF production.

The kindlin 3 pleckstrin homology domain has an essential role in lymphocyte function-associated antigen 1 (LFA-1) integrin-mediated B cell adhesion and migration.

The protein kindlin 3 is mutated in the leukocyte adhesion deficiency III (LAD-III) disorder, leading to widespread infection due to the failure of leukocytes to migrate into infected tissue sites. To gain understanding of how kindlin 3 controls leukocyte function, we have focused on its pleckstrin homology (PH) domain and find that deletion of this domain eliminates the ability of kindlin 3 to participate in adhesion and migration of B cells mediated by the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). PH domains are often involved in membrane localization of proteins through binding to phosphoinositides. We show that the kindlin 3 PH domain has binding affinity for phosphoinositide PI(3,4,5)P3 over PI(4,5)P2. It has a major role in membrane association of kindlin 3 that is enhanced by the binding of LFA-1 to intercellular adhesion molecule 1 (ICAM-1). A splice variant, kindlin 3-IPRR, has a four-residue insert in the PH domain at a critical site that influences phosphoinositide binding by enhancing binding to PI(4,5)P2 as well as by binding to PI(3,4,5)P3. However kindlin 3-IPRR is unable to restore the ability of LAD-III B cells to adhere to and migrate on LFA-1 ligand ICAM-1, potentially by altering the dynamics or PI specificity of binding to the membrane. Thus, the correct functioning of the kindlin 3 PH domain is central to the role that kindlin 3 performs in guiding lymphocyte adhesion and motility behavior, which in turn is required for a successful immune response.

S100A9 has a protective role in inflammation-induced skin carcinogenesis.

The S100A8/A9 heterodimer is expressed by myeloid cells where its function has been extensively investigated. Immune cell S100A8/A9 promotes proinflammatory effects, and its absence is often associated with lack of leukocyte recruitment resulting in protection in terms of disease progression. S100A8/A9 is also expressed by certain epithelia, either constitutively as in mucosal epithelia or following stimulation as in skin keratinocytes. The role of the heterodimer in this context has not been as frequently explored. In this study, the incidence of skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) in S100a9(-/-) mice has been investigated. Unlike the immune disorders and certain models of cancer, absence of S100A8/A9 caused an increased incidence in skin of papillomas and, subsequently, squamous cell carcinomas. Although associated in S100a9(-/-) mice with increased recruitment of neutrophils and T cells, a bone marrow chimera experiment revealed the major defect to be primarily due to the absence of S100A8/A9 in the skin keratinocytes. S100a9(-/-) skin displayed enhanced Ki-67 expression over the time period of appearance of the papillomas suggesting an effect of S100A8/A9 in regulating proliferation in the epidermal layer. Thus, despite immune cell recruitment in S100a9(-/-) mouse skin that might have been predicted to promote tumor growth, it was the absence of S100A8/A9 in skin keratinocytes that dominated in terms of papilloma formation. The study highlights the importance of the S100A8/A9-expressing skin epidermal layer in controlling skin tumor formation and suggests that the influence of the heterodimer is dependent on the tissue context in which it is expressed.

Novel automated tracking analysis of particles subjected to shear flow: kindlin-3 role in B cells.

Shear flow assays are used to mimic the influence of physiological shear force in diverse situations such as leukocyte rolling and arrest on the vasculature, capture of nanoparticles, and bacterial adhesion. Analysis of such assays usually involves manual counting, is labor-intensive, and is subject to bias. We have developed the Leukotrack program that incorporates a novel (to our knowledge) segmentation routine capable of reliable detection of cells in phase contrast images. The program also automatically tracks rolling cells in addition to those that are more firmly attached and migrating in random directions. We demonstrate its use in the analysis of lymphocyte arrest mediated by one or more active conformations of the integrin LFA-1. Activation of LFA-1 is a multistep process that depends on several proteins including kindlin-3, the protein that is mutated in leukocyte adhesion deficiency-III patients. We find that the very first stage of LFA-1-mediated attaching is unable to proceed in the absence of kindlin-3. Our evidence indicates that kindlin-3-mediated high-affinity LFA-1 controls both the early transient integrin-dependent adhesions in addition to the final stable adhesions made under flow conditions.

T lymphocytes orient against the direction of fluid flow during LFA-1-mediated migration.

As they leave the blood stream and travel to lymph nodes or sites of inflammation, T lymphocytes are captured by the endothelium and migrate along the vascular wall to permissive sites of transmigration. These processes take place under the influence of hemodynamic shear stress; therefore, we investigated how migrational speed and directionality are influenced by variations in shear stress. We examined human effector T lymphocytes on intercellular adhesion molecule 1 (ICAM-1)-coated surfaces under the influence of shear stresses from 2 to 60 dyn.cm(-2). T lymphocytes were shown to respond to shear stress application by a rapid (30 s) and fully reversible orientation of their migration against the fluid flow without a change in migration speed. Primary T lymphocytes migrating on ICAM-1 in the presence of uniformly applied SDF-1α were also found to migrate against the direction of shear flow. In sharp contrast, neutrophils migrating in the presence of uniformly applied fMLP and leukemic HSB2 T lymphocytes migrating on ICAM-1 alone oriented their migration downstream, with the direction of fluid flow. Our findings suggest that, in addition to biochemical cues, shear stress is a contributing factor to leukocyte migration directionality.

The integrin LFA-1 signals through ZAP-70 to regulate expression of high-affinity LFA-1 on T lymphocytes.

The integrin lymphocyte function-associated antigen 1 (LFA-1) controls many functions of T lymphocytes and is particularly essential during lymphocyte migration from blood into tissues. LFA-1 is considered to initiate "outside-in" signaling when bound to ligand intercellular adhesion molecule 1 (ICAM-1), but little is known about the proteins involved or where in the cell such LFA-1-mediated signaling might be operating. Here we show that LFA-1 is constitutively associated with the protein tyrosine kinases Lck and zeta chain-associated protein of 70 kDa (ZAP-70). When LFA-1 binds ICAM-1, both kinases become phosphorylated and the consequence of kinase activation is the conversion of intermediate- to high-affinity LFA-1 and an increase in close contact with ICAM-1. In the polarized T lymphocyte, phospho-ZAP-70 is concentrated within a region of high-affinity LFA-1 that includes talin and encompasses the lamella/lamellipodial interface as well as further back in the cell. Deficiency of ZAP-70 through inhibition or knockdown in T lymphocytes decreases the speed of migration on ICAM-1, as well as reducing firm adhesion under shear-flow conditions. Through its control of high-affinity LFA-1, the LFA-1/Lck/ZAP-70 complex is in position to initiate the rapid adhesion strengthening and migration necessary for T-lymphocyte responses when stimulated vasculature is encountered at sites of infection or injury.

G-CSF-mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands.

Emergency mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) is a key event of early cellular immunity. The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) stimulates this process, but it is unknown how individual neutrophils respond in situ. We show by intravital 2-photon microscopy that a systemic dose of human clinical-grade G-CSF rapidly induces the motility and entry of neutrophils into blood vessels within the tibial BM of mice. Simultaneously, the neutrophil-attracting chemokine KC (Cxcl1) spikes in the blood. In mice lacking the KC receptor Cxcr2, G-CSF fails to mobilize neutrophils and antibody blockade of Cxcr2 inhibits the mobilization and induction of neutrophil motility in the BM. KC is expressed by megakaryocytes and endothelial cells in situ and is released in vitro by megakaryocytes isolated directly from BM. This production of KC is strongly increased by thrombopoietin (TPO). Systemic G-CSF rapidly induces the increased production of TPO in BM. Accordingly, a single injection of TPO mobilizes neutrophils with kinetics similar to G-CSF, and mice lacking the TPO receptor show impaired neutrophil mobilization after short-term G-CSF administration. Thus, a network of signaling molecules, chemokines, and cells controls neutrophil release from the BM, and their mobilization involves rapidly induced Cxcr2-mediated motility controlled by TPO as a pacemaker.

The integrins Mac-1 and alpha4beta1 perform crucial roles in neutrophil and T cell recruitment to lungs during Streptococcus pneumoniae infection.

Neutrophils and T cells play an important role in host protection against pulmonary infection caused by Streptococcus pneumoniae. However, the role of the integrins in recruitment of these cells to infected lungs is not well understood. In this study we used the twin approaches of mAb blockade and gene-deficient mice to investigate the relative impact of specific integrins on cellular recruitment and bacterial loads following pneumococcal infection. We find that both Mac-1 (CD11b/CD18) and α(4)ß(1) (CD49d/CD29) integrins, but surprisingly not LFA-1 (CD11a/CD18), contribute to two aspects of the response. In terms of recruitment from the circulation into lungs, neutrophils depend on Mac-1 and α(4)ß(1), whereas the T cells are entirely dependent on α(4)ß(1). Second, immunohistochemistry results indicate that adhesion also plays a role within infected lung tissue itself. There is widespread expression of ICAM-1 within lung tissue. Use of ICAM-1(-/-) mice revealed that neutrophils make use of this Mac-1 ligand, not for lung entry or for migration within lung tissue, but for combating the pneumococcal infection. In contrast to ICAM-1, there is restricted and constitutive expression of the α(4)ß(1) ligand, VCAM-1, on the bronchioles, allowing direct access of the leukocytes to the airways via this integrin at an early stage of pneumococcal infection. Therefore, integrins Mac-1 and α(4)ß(1) have a pivotal role in prevention of pneumococcal outgrowth during disease both in regulating neutrophil and T cell recruitment into infected lungs and by influencing their behavior within the lung tissue itself.

Loss of myeloid related protein-8/14 exacerbates cardiac allograft rejection.

BACKGROUND: The calcium-binding proteins myeloid-related protein (MRP)-8 (S100A8) and MRP-14 (S100A9) form MRP-8/14 heterodimers (S100A8/A9, calprotectin) that regulate myeloid cell function and inflammatory responses and serve as early serum markers for monitoring acute allograft rejection. Despite functioning as a proinflammatory mediator, the pathophysiological role of MRP-8/14 complexes in cardiovascular disease is incompletely defined. This study investigated the role of MRP-8/14 in cardiac allograft rejection using MRP-14(-/-) mice that lack MRP-8/14 complexes. METHODS AND RESULTS: We examined parenchymal rejection after major histocompatibility complex class II allomismatched cardiac transplantation (bm12 donor heart and B6 recipients) in wild-type (WT) and MRP-14(-/-) recipients. Allograft survival averaged 5.9±2.9 weeks (n=10) in MRP-14(-/-) recipients compared with >12 weeks (n=15; P<0.0001) in WT recipients. Two weeks after transplantation, allografts in MRP-14(-/-) recipients had significantly higher parenchymal rejection scores (2.8±0.8; n=8) than did WT recipients (0.8±0.8; n=12; P<0.0001). Compared with WT recipients, allografts in MRP-14(-/-) recipients had significantly increased T-cell and macrophage infiltration and increased mRNA levels of interferon-γ and interferon-γ-associated chemokines (CXCL9, CXCL10, and CXCL11), interleukin-6, and interleukin-17 with significantly higher levels of Th17 cells. MRP-14(-/-) recipients also had significantly more lymphocytes in the adjacent para-aortic lymph nodes than did WT recipients (cells per lymph node: 23.7±0.7×10(5) for MRP-14(-/-) versus 6.0±0.2×10(5) for WT; P<0.0001). The dendritic cells (DCs) of the MRP-14(-/-) recipients of bm12 hearts expressed significantly higher levels of the costimulatory molecules CD80 and CD86 than did those of WT recipients 2 weeks after transplantation. Mixed leukocyte reactions with allo-endothelial cell-primed MRP-14(-/-) DCs resulted in significantly higher antigen-presenting function than reactions using WT DCs. Ovalbumin-primed MRP-14(-/-) DCs augmented proliferation of OT-II (ovalbumin-specific T cell receptor transgenic) CD4(+) T cells with increased interleukin-2 and interferon-γ production. Cardiac allografts of B6 major histocompatibility complex class II(-/-) hosts and of B6 WT hosts receiving MRP-14(-/-) DCs had significantly augmented inflammatory cell infiltration and accelerated allograft rejection compared with WT DCs from transferred recipient allografts. Bone marrow-derived MRP-14(-/-) DCs infected with MRP-8 and MRP-14 retroviral vectors showed significantly decreased CD80 and CD86 expression compared with controls, indicating that MRP-8/14 regulates B7-costimulatory molecule expression. CONCLUSIONS: Our results indicate that MRP-14 regulates B7 molecule expression and reduces antigen presentation by DCs and subsequent T-cell priming. The absence of MRP-14 markedly increased T-cell activation and exacerbated allograft rejection, indicating a previously unrecognized role for MRP-14 in immune cell biology.

Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell.

T lymphocytes use LFA-1 to migrate into lymph nodes and inflammatory sites. To investigate the mechanisms regulating this migration, we utilize mAbs selective for conformational epitopes as probes for active LFA-1. Expression of the KIM127 epitope, but not the 24 epitope, defines the extended conformation of LFA-1, which has intermediate affinity for ligand ICAM-1. A key finding is that KIM127-positive LFA-1 forms new adhesions at the T lymphocyte leading edge. This LFA-1 links to the cytoskeleton through alpha-actinin-1 and disruption at the level of integrin or actin results in loss of cell spreading and migratory speed due to a failure of attachment at the leading edge. The KIM127 pattern contrasts with high-affinity LFA-1 that expresses both 24 and KIM127 epitopes, is restricted to the mid-cell focal zone and controls ICAM-1 attachment. Identification of distinctive roles for intermediate- and high-affinity LFA-1 in T lymphocyte migration provides a biological function for two active conformations of this integrin for the first time.