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Background: The vascular endothelium maintains tissue-fluid homeostasis by controlling the passage of large molecules and fluid between the blood and interstitial space. The interaction of catenins and the actin cytoskeleton with VE-cadherin (vascular endothelial cadherin) is the primary mechanism for stabilizing AJs (adherens junctions), thereby preventing lung vascular barrier disruption. Members of the Rho (Ras homology) family of GTPases and conventional GEFs (guanine exchange factors) of these GTPases have been demonstrated to play important roles in regulating endothelial permeability. Here, we evaluated the role of DOCK4 (dedicator of cytokinesis 4)—an unconventional Rho family GTPase GEF in vascular function. Methods: We generated mice deficient in DOCK4‚ used DOCK4 silencing and reconstitution approaches in human pulmonary artery endothelial cells‚ used assays to evaluate protein localization, endothelial cell permeability, and small GTPase activation. Results: Our data show that DOCK4-deficient mice are viable. However, these mice have hemorrhage selectively in the lung, incomplete smooth muscle cell coverage in pulmonary vessels, increased basal microvascular permeability, and impaired response to S1P (sphingosine-1-phosphate)–induced reversal of thrombin-induced permeability. Consistent with this, DOCK4 rapidly translocates to the cell periphery and associates with the detergent-insoluble fraction following S1P treatment, and its absence prevents S1P-induced Rac-1 activation and enhancement of barrier function. Moreover, DOCK4-silenced pulmonary artery endothelial cells exhibit enhanced basal permeability in vitro that is associated with enhanced Rho GTPase activation. Conclusions: Our findings indicate that DOCK4 maintains AJs necessary for lung vascular barrier function by establishing the normal balance between RhoA (Ras homolog family member A) and Rac-1–mediated actin cytoskeleton remodeling, a previously unappreciated function for the atypical GEF family of molecules. Our studies also identify S1P as a potential upstream regulator of DOCK4 activity.

Classical dendritic cells (cDC) are professional antigen-presenting cells (APC) that regulate immunity and tolerance. Neutrophil-derived cells with properties of DCs (nAPC) are observed in human diseases and after culture of neutrophils with cytokines. Here we show that FcγR-mediated endocytosis of antibody-antigen complexes or an anti-FcγRIIIB-antigen conjugate converts neutrophils into nAPCs that, in contrast to those generated with cytokines alone, activate T cells to levels observed with cDCs and elicit CD8+ T cell-dependent anti-tumor immunity in mice. Single cell transcript analyses and validation studies implicate the transcription factor PU.1 in neutrophil to nAPC conversion. In humans, blood nAPC frequency in lupus patients correlates with disease. Moreover, anti-FcγRIIIB-antigen conjugate treatment induces nAPCs that can activate autologous T cells when using neutrophils from individuals with myeloid neoplasms that harbor neoantigens or those vaccinated against bacterial toxins. Thus, anti-FcγRIIIB-antigen conjugate-induced conversion of neutrophils to immunogenic nAPCs may represent a possible immunotherapy for cancer and infectious diseases., Neutrophils are versatile immune cells that may also serve as antigen-presenting cells (APC). Here the authors show that engaging FcγRs on neutrophils with immune complexes or an anti-FcγR-antigen conjugate induces neutrophil APC with comparable functions as classical dendritic cells, and with therapeutic potentials for cancer and infectious diseases.

Summary Background T cells control viral infection, promote vaccine durability, and in coronavirus disease 2019 (COVID-19) associate with mild disease. We investigated whether prior measles-mumps-rubella (MMR) or tetanus-diphtheria-pertussis (Tdap) vaccination elicits cross-reactive T cells that mitigate COVID-19. Methods Antigen-presenting cells (APC) loaded ex vivo with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), MMR, or Tdap antigens and autologous T cells from COVID-19-convalescent participants, uninfected individuals, and COVID-19 mRNA-vaccinated donors were co-cultured. T cell activation and phenotype were detected by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assays and flow cytometry. ELISAs (enzyme-linked immunosorbant assays) and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and single-cell RNA sequencing (scRNA-seq) identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes. Findings High correlation was observed between T cell responses to SARS-CoV-2 (spike-S1 and nucleocapsid) and MMR and Tdap proteins in COVID-19-convalescent and -vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (effector memory re-expressing CD45RA on T cells [TEMRA]) implicated in protective, anti-viral immunity, and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR, and Tdap epitopes had TEMRA features. Indices of disease severity were reduced in MMR- or Tdap-vaccinated individuals by 32%–38% and 20%–23%, respectively, among COVID-19 patients. Conclusions Tdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19. Funding This study was supported by a National Institutes of Health (R01HL065095, R01AI152522, R01NS097719) donation from Barbara and Amos Hostetter and the Chleck Foundation.

Monocytes undergo phenotypic and functional changes in response to inflammatory cues, but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes, which are preferentially recruited, dwell within glomerular capillaries, and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically, the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells, findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2–activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients, and their frequency correlates with clinical disease. In conclusion, CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2–activated vasculature and may establish a CCR2-based autocrine, feed-forward loop that amplifies renal inflammation.

BACKGROUND T cells control viral infection and promote vaccine durability and in COVID-19 associate with mild disease. We investigated whether prior Measles-Mumps-Rubella (MMR) or Tetanus-Diptheria-Pertussis (Tdap) vaccination elicit cross-reactive T cells that mitigate COVID-19. METHODS Antigen presenting cells (APC) loaded ex vivo with SARS-CoV-2, MMR or Tdap antigens and autologous T cells from COVID-19 convalescent and uninfected individuals, and COVID-19 mRNA vaccinated donors were co-cultured and T cell activation and phenotype were detected by IFN-γ ELISpot assays and flow cytometry. ELISA assays and validation studies identified the APC-derived cytokine(s) driving T cell activation. TCR clonotyping and scRNA-seq identified cross-reactive T cells and their transcriptional profile. A propensity-weighted analysis of COVID-19 patients estimated the effects of MMR and Tdap vaccination on COVID-19 outcomes. FINDINGS High correlation was observed between T cell responses to SARS-CoV-2 (Spike-S1 and Nucleocapsid) and MMR and Tdap proteins in COVID-19 convalescent and vaccinated individuals. The overlapping T cell population contained an effector memory T cell subset (TEMRA) implicated in protective, anti-viral immunity and their detection required APC-derived IL-15, known to sensitize T cells to activation. Cross-reactive TCR repertoires detected in antigen-experienced T cells recognizing SARS-CoV-2, MMR and Tdap epitopes had TEMRA features. Indices of disease severity were reduced in MMR or Tdap vaccinated individuals by 32-38% and 20-23% respectively, among COVID-19 patients. CONCLUSIONS Tdap and MMR memory T cells reactivated by SARS-CoV-2 may provide protection against severe COVID-19 disease. FUNDING National Institutes of Health (R01HL065095, R01AI152522, R01NS097719), donation from Barbara and Amos Hostetter and the Chleck Foundation., Graphical Abstract, T cells critically control infection and effective vaccination. In COVID-19 convalescent or COVID-19 vaccinated individuals, memory T cells previously generated by MMR or Tdap vaccines are reactivated by SARS-CoV-2 antigens and have features of TEMRA, implicated in anti-viral immunity. Prior MMR or Tdap vaccination may protect against severe COVID-19 disease.

SUMMARY The interaction of the human FcγRIIA with immune complexes (ICs) promotes neutrophil activation and thus must be tightly controlled to avoid damage to healthy tissue. Here, we demonstrate that a fungal-derived soluble β-1,3/1,6-glucan binds to the glycosphingolipid long-chain lactosylceramide (LacCer) to reduce FcγRIIA-mediated recruitment to immobilized ICs under flow, a process requiring high-affinity FcγRIIA-immunoglobulin G (IgG) interactions. The inhibition requires Lyn phosphorylation of SHP-1 phosphatase and the FcγRIIA immunotyrosine-activating motif. β-glucan reduces the effective 2D affinity of FcγRIIA for IgG via Lyn and SHP-1 and, in vivo, inhibits FcγRIIA-mediated neutrophil recruitment to intravascular IgG deposited in the kidney glomeruli in a glycosphingolipid- and Lyn-dependent manner. In contrast, β-glucan did not affect FcγR functions that bypass FcγR affinity for IgG. In summary, we have identified a pathway for modulating the 2D affinity of FcγRIIA for ligand that relies on LacCer-Lyn-SHP-1-mediated inhibitory signaling triggered by β-glucan, a previously described activator of innate immunity., In brief Okubo et al. demonstrate that β-glucan binding to the glycosphingolipid lactosylceramide engages a Lyn kinase to SHP-1 phosphatase pathway that reduces FcγRIIA binding propensity for IgG, which suggests FcγRIIA affinity regulation by “inside-out” signaling. The β-glucan-lactosylceramide-Lyn axis prevents FcγRIIA-dependent neutrophil recruitment in vitro and to intravascular IgG deposits following glomerulonephritis., Graphical Abstract

Vascular-deposited IgG immune complexes promote neutrophil recruitment, but how this process is regulated is still unclear. Here we show that the CD18 integrin Mac-1, in its bent state, interacts with the IgG receptor FcγRIIA in cis to reduce the affinity of FcγRIIA for IgG and inhibit FcγRIIA-mediated neutrophil recruitment under flow. The Mac-1 rs1143679 lupus-risk variant reverses Mac-1 inhibition of FcγRIIA, as does a Mac-1 ligand and a mutation in Mac-1’s ligand binding αI-domain. Sialylated complex glycans on FcγRIIA interact with the αI-domain via divalent cations, and this interaction is required for FcγRIIA inhibition by Mac-1. Human neutrophils deficient in CD18 integrins exhibit augmented FcγRIIA-dependent recruitment to IgG-coated endothelium. In mice, CD18 integrins on neutrophils dampen IgG-mediated neutrophil accumulation in the kidney. In summary, cis interaction between sialylated FcγRIIA and the αI-domain of Mac-1 alters the threshold for IgG-mediated neutrophil recruitment. A disruption of this interaction may increase neutrophil influx in autoimmune diseases., Deposited immune complexes (IC) promote neutrophil recruitment, but the fine tuning of this process is still unclear. Here the authors show that the cis interaction of the IC receptor, FcγRIIA and CD18 integrin, Mac-1, on the neutrophil surface modulates neutrophil adhesion, with FcγRIIA sialylation specifically implicated in this interaction.

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe "big heart" and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.

The kidney glomerular capillaries are frequent sites of immune complex deposition and subsequent neutrophil accumulation in post-infectious and rapidly progressive glomerulonephritis. However, the mechanisms of neutrophil recruitment remain enigmatic, and there is no targeted therapeutic to avert this proximal event in glomerular inflammation. The uniquely human activating Fc receptor FcγRIIA promotes glomerular neutrophil accumulation and damage in anti-glomerular basement membrane-induced (anti-GBM-induced) glomerulonephritis when expressed on murine neutrophils. Here, we found that neutrophils are directly captured by immobilized IgG antibodies under physiological flow conditions in vitro through FcγRIIA-dependent, Abl/Src tyrosine kinase-mediated F-actin polymerization. Biophysical measurements showed that the lifetime of FcγRIIA-IgG bonds increased under mechanical force in an F-actin-dependent manner, which could enable the capture of neutrophils under physiological flow. Kidney intravital microscopy revealed that circulating neutrophils, which were similar in diameter to glomerular capillaries, abruptly arrested following anti-GBM antibody deposition via neutrophil FcγRIIA and Abl/Src kinases. Accordingly, inhibition of Abl/Src with bosutinib reduced FcγRIIA-mediated glomerular neutrophil accumulation and renal injury in experimental, crescentic anti-GBM nephritis. These data identify a pathway of neutrophil recruitment within glomerular capillaries following IgG deposition that may be targeted by bosutinib to avert glomerular injury.

SummaryEndothelial-dependent mechanisms of mononuclear cell influx are not well understood. We showed that acute stimulation of murine microvascular endothelial cells expressing the tumor necrosis factor receptors TNFR1 and TNFR2 with the soluble cytokine TNF led to CXCR3 chemokine generation. The TNF receptors signaled through interferon regulatory factor-1 (IRF1) to induce interferon-β (IFN-β) and subsequent autocrine signaling via the type I IFN receptor and the transcription factor STAT1. Both TNFR2 and TNFR1 were required for IRF1-IFNβ signaling and, in human endothelial cells TNFR2 expression alone induced IFN-β signaling and monocyte recruitment. In vivo, TNFR1 was required for acute renal neutrophil and monocyte influx after systemic TNF treatment, whereas the TNFR2-IRF1-IFN-β autocrine loop was essential only for macrophage accumulation. In a chronic model of proliferative nephritis, IRF1 and renal-expressed TNFR2 were essential for sustained macrophage accumulation. Thus, our data identify a pathway in endothelial cells that selectively recruits monocytes during a TNF-induced inflammatory response.

The C-type lectin receptor dectin-1 and the integrin Mac-1 have key roles in controlling fungal infection. Here, we demonstrate that dectin-1- and Mac-1-induced activation of protein kinase Cδ in neutrophils, independent of the Card9 adaptor, is required for reactive oxygen species production and for intracellular killing upon Candida albicans uptake. Protein kinase Cδ was also required for zymosan-induced cytokine generation in neutrophils. In macrophages, protein kinase Cδ deficiency prevented fungi-induced reactive oxygen species generation but had no effect on activation of TGF-β-activated kinase-1, an effector of Card9, or nuclear factor κB activation, nor did it affect phagolysosomal maturation, autophagy, or intracellular C. albicans killing. In vivo, protein kinase Cδ–deficient mice were highly susceptible to C. albicans and Aspergillus fumigatus infection, which was partially rescued with adoptively transferred wild-type neutrophils. Thus, protein kinase Cδ activation downstream of dectin-1 and Mac-1 has an important role in neutrophil, but not macrophage, functions required for host defense against fungal pathogens.

The blood-testis barrier (BTB), formed between adjacent Sertoli cells, undergoes extensive remodeling to facilitate the transport of preleptotene spermatocytes across the barrier from the basal to apical compartments of the seminiferous tubules for further development and maturation into spermatozoa. The actin cytoskeleton serves unique structural and supporting roles in this process, but little is known about the role of microtubules and their regulators during BTB restructuring. The large isoform of the cAMP-responsive scaffold protein AKAP9 regulates microtubule dynamics and nucleation at the Golgi. We found that conditional deletion of Akap9 in mice after the initial formation of the BTB at puberty leads to infertility. Akap9 deletion results in marked alterations in the organization of microtubules in Sertoli cells and a loss of barrier integrity despite a relatively intact, albeit more apically localized F-actin and BTB tight junctional proteins. These changes are accompanied by a loss of haploid spermatids due to impeded meiosis. The barrier, however, progressively reseals in older Akap9 null mice, which correlates with a reduction in germ cell apoptosis and a greater incidence of meiosis. However, spermiogenesis remains defective, suggesting additional roles for AKAP9 in this process. Together, our data suggest that AKAP9 and, by inference, the regulation of the microtubule network are critical for BTB function and subsequent germ cell development during spermatogenesis.

Elevated plasma concentrations of TNF receptors 1 and 2 (TNFR1 and TNFR2) predict development of ESRD in patients with type 2 diabetes without proteinuria, suggesting these markers may contribute to the pathogenesis of renal decline. We investigated whether circulating markers of the TNF pathway determine GFR loss among patients with type 1 diabetes. We followed two cohorts comprising 628 patients with type 1 diabetes, normal renal function, and no proteinuria. Over 12 years, 69 patients developed estimated GFR less than 60 mL/min per 1.73 m 2 (16 per 1000 person-years). Concentrations of TNFR1 and TNFR2 were strongly associated with risk for early renal decline. Renal decline was associated only modestly with total TNFa concentration and appeared unrelated to free TNFa. The cumulative incidence of estimated GFR less than 60 mL/min per 1.73 m 2 for patients in the highest TNFR2 quartile was 60% after 12 years compared with 5%–19% in the remaining quartiles. In Cox proportional hazards analysis, patients with TNFR2 values in the highest quartile were threefold more likely to experience renal decline than patients in the other quartiles (hazard ratio, 3.0; 95% confidence interval, 1.7–5.5). The risk associated with high TNFR1 values was slightly less than that associated with high TNFR2 values. TNFR levels were unrelated to baseline free TNFa level and remained stable over long periods within an individual. In conclusion, early GFR loss in patients with type 1 diabetes without proteinuria is strongly associated with circulating TNF receptor levels but not TNFa levels (free or total).

Levels of proinflammatory cytokines associate with risk for developing type 2 diabetes but whether chronic inflammation contributes to the development of diabetic complications, such as ESRD, is unknown. In the 1990s, we recruited 410 patients with type 2 diabetes for studies of diabetic nephropathy and recorded their characteristics at enrollment. During 12 years of follow-up, 59 patients developed ESRD (17 per 1000 patient-years) and 84 patients died without ESRD (24 per 1000 patient-years). Plasma markers of systemic inflammation, endothelial dysfunction, and the TNF pathway were measured in the study entry samples. Of the examined markers, only TNF receptors 1 and 2 (TNFR1 and TNFR2) associated with risk for ESRD. These two markers were highly correlated, but ESRD associated more strongly with TNFR1. The cumulative incidence of ESRD for patients in the highest TNFR1 quartile was 54% after 12 years but only 3% for the other quartiles (P

SummaryResistance to fungal infections is attributed to engagement of host pattern-recognition receptors, notably the β-glucan receptor Dectin-1 and the integrin Mac-1, which induce phagocytosis and antifungal immunity. However, the mechanisms by which these receptors coordinate fungal clearance are unknown. We show that upon ligand binding, Dectin-1 activates Mac-1 to also recognize fungal components, and this stepwise process is critical for neutrophil cytotoxic responses. Both Mac-1 activation and Dectin-1- and Mac-1-induced neutrophil effector functions require Vav1 and Vav3, exchange factors for RhoGTPases. Mac-1- or Vav1,3-deficient mice have increased susceptibility to systemic candidiasis that is not due to impaired neutrophil recruitment but defective intracellular killing of C. albicans yeast forms, and Mac-1 or Vav1,3 reconstitution in hematopoietic cells restores resistance. Our results demonstrate that antifungal immunity depends on Dectin-1-induced activation of Mac-1 functions that is coordinated by Vav proteins, a pathway that may localize cytotoxic responses of circulating neutrophils to infected tissues.

Adhesive forces at endothelial cell-cell borders maintain vascular integrity. cAMP enhances barrier properties and controls cellular processes through protein kinase A bound to A-kinase anchoring proteins (AKAPs). It also activates exchange protein directly activated by cAMP (Epac1), an exchange factor for Ras-related protein 1 (Rap1) GTPases that promotes cadherin- and integrin-mediated adhesion through effects on the actin cytoskeleton. We demonstrate that AKAP9 facilitates the microtubule polymerization rate in endothelial cells, interacts with Epac1, and is required for Epac1-stimulated microtubule growth. AKAP9 is not required for maintaining barrier properties under steady-state conditions. Rather, it is essential when the cell is challenged to make new adhesive contacts, as is the case when Epac activation enhances barrier function through a mechanism that, surprisingly, requires integrin adhesion at cell-cell contacts. In the present study, defects in Epac-induced responses in AKAP9-silenced cells were evident despite an intact Epac-induced increase in Rap activation, cortical actin, and vascular endothelial-cadherin adhesion. We describe a pathway that integrates Epac-mediated signals with AKAP9-dependent microtubule dynamics to coordinate integrins at lateral borders.

The mechanisms driving T cell homing to lymph nodes and migration to tissue are well described but little is known about factors that affect T cell egress from tissues. Here, we generate mice with a T cell-specific deletion of the scaffold protein A kinase anchoring protein 9 (AKAP9) and use models of inflammatory disease to demonstrate that AKAP9 is dispensable for T cell priming and migration into tissues and lymph nodes, but is required for T cell retention in tissues. AKAP9 deficiency results in increased T cell egress to draining lymph nodes, which is associated with impaired T cell re-activation in tissues and protection from organ damage. AKAP9-deficient T cells exhibit reduced microtubule-dependent recycling of TCRs back to the cell surface and this affects antigen-dependent activation, primarily by non-classical antigen-presenting cells. Thus, AKAP9-dependent TCR trafficking drives efficient T cell re-activation and extends their retention at sites of inflammation with implications for disease pathogenesis., A-kinase anchoring protein 9 (AKAP9) is a scaffold protein that binds signalling proteins and regulates microtubules. Here the authors show that during inflammation AKAP9 in T cells is required for their reactivation and retention at the inflammation site and that its deletion protects from inflammation-induced organ damage.

Endothelial cell-cell junctional proteins and cortical actin are of central importance for regulating vascular permeability. Rap1, a member of the Ras family of GTPases, is enriched at endothelial cell-cell contacts and activated by cyclic AMP (cAMP) through a PKA-independent pathway. Activation of a cAMP-inducible guanine-exchange factor for Rap, Epac, results in markedly enhanced basal endothelial barrier function by increasing cortical actin and subsequent redistribution of adherens and tight junctional molecules to cell-cell contacts. Activation of Epac also counteracts thrombin-induced hyperpermeability through down-regulation of Rho GTPase activation, suggesting cross-talk between Rap and Rho GT-Pases. Thus, Epac/Rap activation represents a new pathway for regulating endothelial cell barrier function.

Phagocytosis of complement-opsonized targets is a primary function of neutrophils at sites of inflammation, and the clearance of neutrophils that have phagocytosed microbes is important for the resolution of inflammation. Our previous work suggests that phagocytosis leads to rapid neutrophil apoptosis that is inhibited by antibody to the beta2 integrin, Mac-1, and requires NADPH oxidase-derived reactive oxygen species (ROS) generated during phagocytosis. Here we report that phagocytosis-induced cell death (PICD) does not occur in Mac-1-deficient murine neutrophils, suggesting that PICD proceeds through a bona fide Mac-1-dependent pathway. A sustained, intracellular oxidative burst is associated with PICD. Furthermore, PICD does not require traditional death receptors, Fas, or tumor necrosis factor (TNF) receptor. TNF but not Fas synergizes with phagocytosis to enhance significantly PICD by increasing the oxidative burst, and this is Mac-1-dependent. Phagocytosis-induced ROS promote cleavage/activation of caspases 8 and 3, key players in most extrinsic ("death receptor") mediated pathways of apoptosis, and caspases 8 and 3 but not caspase 9/mitochondria, are required for PICD. This suggests that ROS target the extrinsic versus the intrinsic ("stress stimulus") apoptotic pathway. Phagocytosis also triggers a competing MAPK/ERK-dependent survival pathway that provides resistance to PICD likely by down-regulating caspase 8 activation. The anti-apoptotic factor granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly enhances ROS generation associated with phagocytosis. Despite this, it completely suppresses PICD by sustaining ERK activation and inhibiting caspase 8 activation in phagocytosing neutrophils. Together, these studies suggest that Mac-1-mediated phagocytosis promotes apoptosis through a caspase 8/3-dependent pathway that is modulated by NADPH oxidase-generated ROS and MAPK/ERK. Moreover, TNF and GM-CSF, likely encountered by phagocytosing neutrophils at inflammatory sites, exploit pro-(ROS) and anti-apoptotic (ERK) signals triggered by phagocytosis to promote or suppress PICD, respectively, and thus modulate the fate of phagocytosing neutrophils.

SummaryLeukocyte CD18 integrins increase their affinity for ligand by transmitting allosteric signals to and from their ligand-binding αI domain. Mechanical forces induce allosteric changes that paradoxically slow dissociation by increasing the integrin/ligand bond lifetimes, referred to as catch bonds. Mac-1 formed catch bonds with its ligands. However, a Mac-1 gene (ITGAM) coding variant (rs1143679, R77H), which is located in the β-propeller domain and is significantly associated with systemic lupus erythematosus risk, exhibits a marked impairment in 2D ligand affinity and affinity maturation under mechanical force. Targeted mutations and activating antibodies reveal that the failure in Mac-1 R77H allostery is rescued by induction of cytoplasmic tail separation and full integrin extension. These findings demonstrate roles for R77, and the β-propeller in which it resides, in force-induced allostery relay and integrin bond stabilization. Defects in these processes may have pathological consequences, as the Mac-1 R77H variant is associated with increased susceptibility to lupus.

© 2014 by Annual Reviews. All rights reserved. Neutrophils and neutrophil-like cells are the major pathogen-fighting immune cells in organisms ranging from slime molds to mammals. Central to their function is their ability to be recruited to sites of infection, to recognize and phagocytose microbes, and then to kill pathogens through a combination of cytotoxic mechanisms. These include the production of reactive oxygen species, the release of antimicrobial peptides, and the recently discovered expulsion of their nuclear contents to form neutrophil extracellular traps. Here we discuss these primordial neutrophil functions, which also play key roles in tissue injury, by providing details of neutrophil cytotoxic functions and congenital disorders of neutrophils. In addition, we present more recent evidence that interactions between neutrophils and adaptive immune cells establish a feed-forward mechanism that amplifies pathologic inflammation. These newly appreciated contributions of neutrophils are described in the setting of several inflammatory and autoimmune diseases.

Neutrophil accumulation is a hallmark of immune complex-mediated inflammatory disorders. Current models of neutrophil recruitment envision the capture of circulating neutrophils by activated endothelial cells. We now demonstrate that immobilized immune complexes alone support the rapid attachment of neutrophils, under physiologic flow conditions. Initial cell tethering requires the low-affinity Fc gamma receptor IIIB (Fc gamma RIIIB), and the beta(2) integrins are additionally required for the subsequent shear-resistant adhesion. The attachment function of Fc gamma RIIIB may be facilitated by its observed presentation on neutrophil microvilli. In vivo, in a model of acute antiglomerular basement membrane nephritis in which immune complexes are accessible to circulating neutrophils, Fc gamma RIII-deficient mice had a significant reduction in neutrophil recruitment. Thus, the interaction of immune complexes with Fc gamma RIII may mediate early neutrophil recruitment in immune complex-mediated inflammation.

Objective Rheumatoid arthritis culminates in joint destruction that, in mouse models of disease, is supported by innate immune molecules, including Fcγ receptors (FcγR) and complement. However, these findings may not be predictive of the outcome in humans, given the structural differences between murine and human activating FcγR on neutrophils, a prominent component of joint exudates. The aim of this study was to examine the role of human neutrophil FcγRIIa in the development of arthritis and probe the underlying mechanism by which FcγRIIa initiates disease. Methods K/BxN mouse serum transfer–induced arthritis was examined in mice expressing human FcγRIIa on neutrophils but lacking their own activating FcγR (γ-chain–deficient mice). The role of mast cells, complement (C3 and C5a), and CD18 integrins in FcγRIIa-initiated disease was examined using cell reconstitution approaches, inhibitors, and functional blocking antibodies, respectively. Crosstalk between the complement receptor C5aR and FcγRIIa on neutrophils was evaluated in vitro. Results The expression of human FcγRIIa on neutrophils was sufficient to restore susceptibility to K/BxN serum–induced neutrophil recruitment, synovitis, and bone destruction in γ-chain–deficient mice. Joint inflammation was robust and proceeded even in the absence of mast cells and vascular permeability, features shown to contribute to disease in wild-type mice. Neutrophil recruitment was dependent on the presence of a CD18 integrin, lymphocyte function–associated antigen 1, and C5aR. In addition, C5aR significantly enhanced FcγRIIa-mediated phagocytosis and oxidative burst in vitro. Conclusion Human and murine activating FcγR on neutrophils are not functionally equivalent, and in humans, they may play a primary role in arthritis. Crosstalk between neutrophil C5aR and FcγRIIa is essential for disease progression, thus highlighting a new aspect of complement during the effector phase of inflammatory arthritis.

The signals linking neutrophil opsonic receptors, FcγRs and complement receptor 3 (Mac-1) to cellular cytotoxic responses are poorly understood. Furthermore, because a deficiency in activating FcγRs reduces both IgG-mediated neutrophil recruitment and tissue injury, the role of FcγRs specifically in mediating neutrophil cytotoxicity in vivo remains unclear. In this study, we demonstrate that neutrophil Vav 1 and 3, guanine exchange factors for Rac GTPases, are required for IgG/FcγR-mediated hemorrhage and edema in the reverse passive Arthus in the lung and skin. Rac GTPases are also required for development of the reverse passive Arthus reaction. A deficiency in Vav 1 and 3 does not affect neutrophil accumulation at the site of immune complex deposition, thus uncoupling neutrophil recruitment and tissue injury. Surprisingly, Vav and Rac proteins are dispensable for the development of the local Shwartzman reaction in vivo and phagocytosis of complement-opsonized RBC in vitro, processes strictly dependent on Mac-1 and complement C3. Thus, FcγR signaling through the Vav and Rac proteins in neutrophils is critical for stimulating immune complex disease while Vav- and Rac-independent pathways promote Mac-1/complement C3-dependent functions.

Tissue-specific silencing of genes may be used for genetic engineering in mice and has possible therapeutic applications in humans. Current strategies in mice rely on Cre/loxP technology requiring the generation of multiple transgenic lines and breeding strategies. Here, we describe the selective silencing of CD18, a leukocyte-specific integrin in neutrophils using a micro RNA (miRNA) strategy that requires the generation of one transgenic line. CD18-specific miRNA hairpin driven by the myeloid specific human MRP8 promoter resulted in the generation of transgenic lines with 75% to 95% reduction in CD18 protein levels in neutrophils and monocytes. Minimal decreases in T cells and a partial diminution in macrophages were observed. Neutrophil CD18 silencing resulted in neutrophilia, splenomegaly, and significant defects in neutrophil trafficking with the degree of alterations correlating with the extent of CD18 silencing. Thus, our data demonstrate the utility of using miRNA approaches to silence genes in neutrophils, which are terminally differentiated cells with a short half-life that largely precludes their genetic manipulation in vitro. Furthermore, the mouse models provide a valuable tool to examine the contribution of CD18 on neutrophils to leukocyte adhesion deficiency type I (LAD-I), a complex inherited disorder in which reduced or absent CD18 expression in multiple leukocyte subsets leads to impaired innate and adaptive immune responses.

Regulated adhesion of T cells by the integrins LFA-1 (lymphocyte function-associated antigen-1) and VLA-4 (very late antigen-4) is essential for T-cell trafficking. The small GTPase Rap1 is a critical activator of both integrins in murine lymphocytes and T-cell lines. Here we examined the contribution of the Rap1 regulatory pathway in integrin activation in primary CD3+ human T cells. We demonstrate that inactivation of Rap1 GTPase in human T cells by expression of SPA1 or Rap1GAP blocked stromal cell-derived factor-1α (SDF-1α)–stimulated LFA-1–ICAM-1 (intercellular adhesion molecule-1) interactions and LFA-1 affinity modulation but unexpectedly did not significantly affect binding of VLA-4 to its ligand VCAM-1 (vascular cell adhesion molecule 1). Importantly, silencing of the Rap1 guanine exchange factor CalDAG-GEFI inhibited SDF-1α- and phorbol 12-myristate 13-acetate (PMA)–induced adhesion to ICAM-1 while having no effect on adhesion to VCAM-1. Pharmacologic inhibition of Phospholipase C (PLC) blocked Rap1 activation and inhibited cell adhesion and polarization on ICAM-1 and VCAM-1. Protein kinase C (PKC) inhibition led to enhanced levels of active Rap1 concomitantly with increased T-cell binding to ICAM-1, whereas adhesion to VCAM-1 was reduced. Thus, PLC/CalDAG-GEFI regulation of Rap1 is selectively required for chemokine- and PMA-induced LFA-1 activation in human T cells, whereas alternate PLC- and PKC-dependent mechanisms are involved in the regulation of VLA-4.

Beta2 integrins are leukocyte-specific membrane receptors that are crucial for host defense. They are best known for promoting neutrophil recruitment into inflamed tissue and pathogen phagocytosis. More recent data suggest that they also modulate neutrophil apoptosis. Neutrophils are terminally differentiated cells, which undergo constitutive apoptosis, and their apoptosis and clearance is required for the resolution of inflammation. Engagement of the beta2 integrin Mac-1 through its adhesion to its ligands, intercellular adhesion molecule-1 (ICAM-1) and fibrinogen, signals survival cues in neutrophils. However, in the presence of pro-apoptotic signals, such as tumor necrosis factor (TNF), Mac-1 engagement accelerates apoptosis. Furthermore, Mac-1-dependent phagocytosis of complement-opsonized pathogens triggers rapid neutrophil apoptosis, which is dependent on NADPH oxidase-generated reactive oxygen species and caspase activation. This is also associated with changes in the transcription profiles of pro- and anti-apoptotic genes. In this review, the beta2 integrin-dependent mechanisms that modulate the decision between life and death in neutrophils are overviewed.

Integrin regulation of neutrophils is essential for appropriate adhesion and transmigration into tissues. Vav proteins are Rho family guanine nucleotide exchange factors that become tyrosine phosphorylated in response to adhesion. Using Vav1/Vav3-deficient neutrophils (Vav1/3ko), we show that Vav proteins are required for multiple β2 integrin-dependent functions, including sustained adhesion, spreading, and complement-mediated phagocytosis. These defects are not attributable to a lack of initial β2 activation as Vav1/3ko neutrophils undergo chemoattractant-induced arrest on intercellular adhesion molecule-1 under flow. Accordingly, in vivo, Vav1/3ko leukocytes arrest on venular endothelium yet are unable to sustain adherence. Thus, Vav proteins are specifically required for stable adhesion. β2-induced activation of Cdc42, Rac1, and RhoA is defective in Vav1/3ko neutrophils, and phosphorylation of Pyk2, paxillin, and Akt is also significantly reduced. In contrast, Vav proteins are largely dispensable for G protein-coupled receptor–induced signaling events and chemotaxis. Thus, Vav proteins play an essential role coupling β2 to Rho GTPases and regulating multiple integrin-induced events important in leukocyte adhesion and phagocytosis.

Phagocytosis of complement-opsonized targets is a primary function of neutrophils at sites of inflammation, and the clearance of neutrophils that have phagocytosed microbes is important for the resolution of inflammation. Our previous work suggests that phagocytosis leads to rapid neutrophil apoptosis that is inhibited by antibody to the beta2 integrin, Mac-1, and requires NADPH oxidase-derived reactive oxygen species (ROS) generated during phagocytosis. Here we report that phagocytosis-induced cell death (PICD) does not occur in Mac-1-deficient murine neutrophils, suggesting that PICD proceeds through a bona fide Mac-1-dependent pathway. A sustained, intracellular oxidative burst is associated with PICD. Furthermore, PICD does not require traditional death receptors, Fas, or tumor necrosis factor (TNF) receptor. TNF but not Fas synergizes with phagocytosis to enhance significantly PICD by increasing the oxidative burst, and this is Mac-1-dependent. Phagocytosis-induced ROS promote cleavage/activation of caspases 8 and 3, key players in most extrinsic ("death receptor") mediated pathways of apoptosis, and caspases 8 and 3 but not caspase 9/mitochondria, are required for PICD. This suggests that ROS target the extrinsic versus the intrinsic ("stress stimulus") apoptotic pathway. Phagocytosis also triggers a competing MAPK/ERK-dependent survival pathway that provides resistance to PICD likely by down-regulating caspase 8 activation. The anti-apoptotic factor granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly enhances ROS generation associated with phagocytosis. Despite this, it completely suppresses PICD by sustaining ERK activation and inhibiting caspase 8 activation in phagocytosing neutrophils. Together, these studies suggest that Mac-1-mediated phagocytosis promotes apoptosis through a caspase 8/3-dependent pathway that is modulated by NADPH oxidase-generated ROS and MAPK/ERK. Moreover, TNF and GM-CSF, likely encountered by phagocytosing neutrophils at inflammatory sites, exploit pro-(ROS) and anti-apoptotic (ERK) signals triggered by phagocytosis to promote or suppress PICD, respectively, and thus modulate the fate of phagocytosing neutrophils.

Phagocytes generate reactive oxygen species, the regulation of which is important in eliminating ingested microbes while limiting tissue damage. Clustering of FcγRs results in the activation of Vav proteins, Rho/Rac guanine nucleotide exchange factors, and results in robust superoxide generation through the NADPH oxidase. In this study, studies in neutrophils isolated from mice deficient in Vav or Rac isoforms demonstrate a critical role for Vav3 in Rac2-dependent activation of the NADPH oxidase following FcγR clustering. However, studies in cytokine-primed cells revealed a strict requirement for Vav1 and Vav3 and Rac1 and Rac2 in the FcγR-mediated oxidative burst. In comparison, Vav was not essential for PMA or G protein-coupled receptor-mediated superoxide generation. The FcγR-mediated oxidative burst defect in Vav-deficient cells was linked to aberrant Rac activation as well as Rac- and actin-polymerization-independent, but PI3K-dependent, phosphorylation of the NADPH oxidase component p40(phox). In macrophages, Vav regulation of Rac GTPases was required specifically in FcγR-mediated activation of the oxidative burst, but not in phagocytosis. Thus, Vav proteins specifically couple FcγR signaling to NADPH oxidase function through a Rac-dependent as well as an unexpected Rac-independent signal that is proximal to NADPH oxidase activation and does not require actin polymerization.