The cytokine midkine supports neutrophil trafficking during acute inflammation by promoting adhesion via ß2 integrins (CD11/CD18).

Emerging evidence suggests a role of the cytokine midkine (MK) in inflammation. In this study, its functional relevance for recruitment of polymorphonuclear neutrophils (PMNs) during acute inflammation was investigated. Intravital microscopy and histologic analysis of tumor necrosis factor-α-stimulated cremaster muscle venules revealed severely compromised leukocyte adhesion and extravasation in MK(-/-) mice compared with MK(+/+) animals. Systemic administration of recombinant MK completely rescued the adhesion defect in MK(-/-) mice. In a hind limb ischemia model, leukocyte accumulation in MK(-/-) mice was significantly diminished compared with MK(+/+) animals. However, MK did not lead to an inflammatory activation of PMNs or endothelial cells suggesting that it does not serve as classical proinflammatory cytokine. Unexpectedly, immobilized MK mediated PMN adhesion under static and flow conditions, whereas PMN-derived MK was dispensable for the induction of adhesion. Furthermore, adhesion strengthening remained unaffected by MK. Flow cytometry revealed that immobilized, but not soluble MK, significantly promoted the high affinity conformation of ß2 integrins of PMNs. Blocking studies of low-density lipoprotein receptor-related protein 1 (LRP1) suggested that LRP1 may act as a receptor for MK on PMNs. Thus, MK seems to support PMN adhesion by promoting the high affinity conformation of ß2 integrins, thereby facilitating PMN trafficking during acute inflammation.

Hematopoietic progenitor kinase 1 (HPK1) is required for LFA-1-mediated neutrophil recruitment during the acute inflammatory response.

Recruitment of polymorphonuclear neutrophils (PMNs) to sites of acute inflammation critically depends on ß2 integrins (CD11/CD18). Recently, the mammalian actin-binding protein 1 (mAbp1) was identified as an important adaptor protein regulating PMN trafficking downstream of ß2 integrins. Here, we show that mAbp1 constitutively co-immunoprecipitated with hematopoietic progenitor kinase 1 (HPK1) in neutrophil-like differentiated HL-60 (dHL-60) cells. HPK1 was enriched at the lamellipodium of polarized dHL-60 cells, where it colocalized with mAbp1 and actin. Functional analysis of PMNs from HPK1-deficient mice showed that HPK1 was critical for CXCL1-induced lymphocyte function-associated antigen 1 (LFA-1)-mediated PMN adhesion to ICAM-1 under flow conditions. Accordingly, CXCL1-mediated induction of high-affinity LFA-1 required HPK1, but macrophage antigen 1 (Mac-1) affinity regulation was independent of HPK1. Intravital microscopy of the mouse cremaster muscle confirmed the defect of CXCL1-induced leukocyte adhesion in HPK1-deficient mice. Furthermore, ß2 integrin-mediated post-adhesion processes-adhesion strengthening, spreading, and directed mechanotactic crawling of PMNs under flow conditions-involved HPK1 in vitro and in vivo. Upon intrascrotal administration of tumor necrosis factor α (TNF-α), PMN adhesion and extravasation were severely compromised in HPK1-deficient mice. In summary, our results indicate that HPK1 is critically involved in LFA-1-mediated PMN trafficking during acute inflammation.

The mammalian actin-binding protein 1 is critical for spreading and intraluminal crawling of neutrophils under flow conditions.

Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was identified as a novel component of the ß(2) integrin-mediated signaling cascade during complement-mediated phagocytosis and firm adhesion of polymorphonuclear neutrophils (PMN) under physiological shear stress conditions. In this study, we found that the genetic ablation of mAbp1 severely compromised not only the induction of adhesion, but also subsequent spreading of leukocytes to the endothelium as assessed by intravital microscopy of inflamed vessels of the cremaster muscle of mice. In vitro studies using murine PMN confirmed that mAbp1 was required for ß(2) integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon ß(2) integrin-mediated adhesion and chemotactic migration of human neutrophil-like differentiated HL-60 cells, mAbp1 was enriched at the leading edge of the polarized cell. Total internal reflection fluorescence microscopy revealed that mAbp1 formed propagating waves toward the front of the lamellipodium, which are characteristic for dynamic reorganization of the cytoskeleton. Accordingly, binding of mAbp1 to actin was increased upon ß(2) integrin-mediated adhesion, as shown by coimmunoprecipitation experiments. However, chemotactic migration under static conditions was unaffected in the absence of mAbp1. In contrast, the downregulation of mAbp1 by RNA interference technique in neutrophil-like differentiated HL-60 cells or the genetic ablation of mAbp1 in leukocytes led to defective migration under flow conditions in vitro and in inflamed cremaster muscle venules in the situation in vivo. In conclusion, mAbp1 is of fundamental importance for spreading and migration under shear stress conditions, which are critical prerequisites for efficient PMN extravasation during inflammation.

Neutrophil functions and autoimmune arthritis in the absence of p190RhoGAP: generation and analysis of a novel null mutation in mice.

Beta(2) integrins of neutrophils play a critical role in innate immune defense, but they also participate in tissue destruction during autoimmune inflammation. p190RhoGAP (ArhGAP35), a regulator of Rho family small GTPases, is required for integrin signal transduction in fibroblasts. Prior studies have also suggested a role for p190RhoGAP in beta(2) integrin signaling in neutrophils. To directly test that possibility, we have generated a novel targeted mutation completely disrupting the p190RhoGAP-encoding gene in mice. p190RhoGAP deficiency led to perinatal lethality and defective neural development, precluding the analysis of neutrophil functions in adult p190RhoGAP(-/-) animals. This was overcome by transplantation of fetal liver cells from p190RhoGAP(-/-) fetuses into lethally irradiated wild-type recipients. Neutrophils from such p190RhoGAP(-/-) bone marrow chimeras developed normally and expressed normal levels of various cell surface receptors. Although p190RhoGAP(-/-) neutrophils showed moderate reduction of beta(2) integrin-mediated adherent activation, they showed mostly normal migration in beta(2) integrin-dependent in vitro and in vivo assays and normal beta(2) integrin-mediated killing of serum-opsonized Staphylococcus aureus and Escherichia coli. A neutrophil- and beta(2) integrin-dependent transgenic model of the effector phase of autoimmune arthritis also proceeded normally in p190RhoGAP(-/-) bone marrow chimeras. In contrast, all the above responses were completely blocked in CD18(-/-) neutrophils or CD18(-/-) bone marrow chimeras. These results suggest that p190RhoGAP likely does not play a major indispensable role in beta(2) integrin-mediated in vitro and in vivo neutrophil functions or the effector phase of experimental autoimmune arthritis.