Jonathan S. Reichner

Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice

The anti‐granulocyte receptor‐1 (Gr‐1) mAb, RB6‐8C5, has been used extensively to deplete neutrophils in mice and to investigate the role of these cells in host defense. RB6‐8C5 binds to Ly6G, which is present on neutrophils, and to Ly6C, which is expressed on neutrophils, dendritic cells, and subpopulations of lymphocytes and monocytes. It is thus likely that in vivo administration of RB6‐8C5 may deplete not only neutrophils but also other Gr‐l+ (Ly6C+) cells. This study describes the use of an Ly6G‐specific mAb, 1A8, as an alternative means to deplete neutrophils. In vivo administration of RB6‐8C5 reduced blood neutrophils and Gr‐1+ monocytes, whereas administration of 1A8 reduced blood neutrophils but not Gr‐1+ monocytes. Plasma TNF‐α in endotoxemia was increased 20‐fold by RB6‐8C5 pretreatment and fourfold by 1A8 pretreatment. In a wound model, pretreatment with either antibody decreased wound neutrophils and macrophages. TNF‐α staining in brefeldin‐treated wound leukocytes was increased by pretreatment with RB6‐8C5, but not 1A8. Neutrophil depletion with 1A8 offers advantages over the use of RB6‐8C5, as it preserves non‐neutrophil Gr‐1+ cells depleted by the anti‐Gr‐1 antibody. The loss of non‐neutrophil Gr‐1+ populations in RB6‐8C5‐treated animals is associated with increased TNF‐α responses, suggesting these cells may function to suppress TNF‐α production.

The phenotype of murine wound macrophages

The phenotype of wound macrophages has not been studied by direct examination of these cells, yet macrophages recruited to sites of injury are described as alternatively activated macrophages, requiring IL‐4 or IL‐13 for phenotypic expression. This study characterized wound macrophage phenotype in the PVA sponge wound model in mice. Eighty‐five percent of wound macrophages isolated 1 day after injury expressed Gr‐1, but only 20% of those isolated at 7 days expressed this antigen. Macrophages from 1‐, 3‐, and 7‐day wounds expressed markers of alternative activation, including mannose receptor, dectin‐1, arginase 1, and Ym1, but did not contain iNOS. Day 1 wound macrophages produced more TNF‐α, more IL‐6, and less TGF‐β than Day 7 wound macrophages. Wound macrophages did not produce IL‐10. The cytokines considered necessary for alternative activation of macrophages, IL‐4 and IL‐13, were not detected in the wound environment and were not produced by wound cells. Wound macrophages did not contain PStat6. Wound fluids inhibited IL‐13‐dependent phosphorylation of Stat6 and contained IL‐13Rα2, a soluble decoy receptor for IL‐13. The phenotype of wound macrophages was not altered in mice lacking IL‐4Rα, which is required for Stat6‐dependent signaling of IL‐4 and IL‐13. Wound macrophages exhibit a complex phenotype, which includes traits associated with alternative and classical activation and changes as the wound matures. The wound macrophage phenotype does not require IL‐4 or IL‐13.

Role of nitric oxide in mediation of macrophage cytotoxicity and apoptosis

Macrophages can recognize and eliminate tumor cells. To this effect, these cells use a variety of cytotoxic effectors. Recent work has paid particular attention to nitric oxide (NO) and its metabolic by-products in mediating macrophage tumor cytotoxicity. Moreover, work from this and other laboratories have indicated that macrophage-dependent, NO mediated tumor cell death meets the morphologic and molecular criteria that define apoptotic cell death. This review will initially discusss the characteristics of macrophage tumor cytotoxicity and the potential mechanisms by which NO can induce apoptosis in tumor cells. In addition, observations of spontaneous and acquired resistance to NO will be analyzed. Lastly, the relevance of results obtained using animal cells to the biology of the human macrophage will be considered.

Distinct arginase isoforms expressed in primary and transformed macrophages : regulation by oxygen tension

Experiments were performed to identify arginase isoforms expressed in primary and transformed rodent macrophages and to determine the molecular mechanisms for the previously observed increase in arginase activity in macrophages cultured in hypoxia or anoxia. Results demonstrate the following: 1) mRNA and protein for hepatic-type AI arginase are expressed in primary cultures of rat and mouse peritoneal macrophages and are enhanced seven- and ninefold, respectively, by lipopolysaccharide (LPS). 2) mRNA for extrahepatic-type AII arginase is constitutively expressed in mouse, but not rat, peritoneal macrophages and is detected in RAW264.7 cells after LPS treatment; neither J774A.1 nor P388D1 cells contain arginase mRNA. 3) AI arginase mRNA, arginase activity in cell lysates, andl-arginine flux through arginase in intact cells are all increased in rat wound-derived and mouse peritoneal macrophages by hypoxic or anoxic culture; AII arginase mRNA is, in contrast, suppressed >50% by O2deprivation. 4) Expression of thel-arginine transporter mCAT-2 is increased greater than twofold by reduced O2 culture. These results demonstrate substantial variability in arginase isoform expression among primary and transformed rodent macrophages. They also identify AI and AII arginase and the mCAT-2 l-arginine transporter as O2-regulated genes.

An Extracellular Matrix–Based Mechanism of Rapid Neutrophil Extracellular Trap Formation in Response to Candida albicans

The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as neutrophil extracellular traps (NETs). The receptor/ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Because the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrix, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern β-glucan by human neutrophils causes rapid (≤30 min) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized β-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn with β-glucan is robust, accounting for 17.2 ± 3.4% of total DNA in the cell population. Release is dependent on β-glucan recognition by complement receptor 3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on β-glucan recognition by complement receptor 3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid antifungal response.

Macrophage-Induced Neutrophil Apoptosis

Macrophages (Mφ) contribute to the resolution of early inflammation by recognizing and ingesting apoptotic polymorphonuclear neutrophils (PMN). In addition, experiments reported here demonstrated that Mφ can actively induce PMN apoptosis. Coculture of cells from 2- or 5-day-old wounds in rats, or of Mφ purified from such preparations, with PMN-rich wound cell populations obtained 1 day after wounding increased PMN apoptosis by >3-fold. Neither resident- nor Proprionibacterium acnes-elicited peritoneal Mφ-induced PMN apoptosis. Apoptosis was not mediated by a soluble factor and required E:T contact. Fixed wound-Mφ and membrane isolates from viable Mφ were as effective as intact cells in inducing PMN apoptosis. Mφ-induced apoptosis was inhibited by peptide Arg-Gly-Asp-Ser, anti-β3 (CD61) Ab, CD36 peptide, or anti-TNF-α Ab. Soluble TNF-α did not induce PMN apoptosis. In additional studies, K562 cells (negative for β3, TNF-α, and Fas ligand) transfected to express either αvβ3 integrin, an uncleavable membrane form of TNF-α, or both were used in cocultures with wound PMN. Only the double transfectants were able to induce PMN apoptosis, an effect inhibited by anti-β3 (CD61) or anti-TNF-α Abs. These results demonstrate that wound Mφ induce PMN apoptosis through a constitutive effector mechanism requiring both intercellular binding through integrin-ligand interactions and membrane-bound TNF-α.

Neutrophil morphology and migration are affected by substrate elasticity.

To reach sites of inflammation, neutrophils execute a series of adhesion and migration events that include transmigration through the vascular endothelium and chemotaxis through the vicinal extracellular matrix until contact is made with the point of injury or infection. These in vivo microenvironments differ in their mechanical properties. Using polyacrylamide gels of physiologically relevant elasticity in the range of 5 to 100 kPa and coated with fibronectin, we tested how neutrophil adhesion, spreading, and migration were affected by substrate stiffness. Neutrophils on the softest gels showed only small changes in spread area, whereas on the stiffest gels they showed a 3-fold increase. During adhesion and migration, the magnitudes of the distortions induced in the gel substrate were independent of substrate stiffness, corresponding to the generation of significantly larger traction stresses on the stiffer gels. Cells migrated more slowly but more persistently on stiffer substrates, which resulted in neutrophils moving greater distances over time despite their slower speeds. The largest tractions were localized to the posterior of migrating neutrophils and were independent of substrate stiffness. Finally, the phosphatidylinositol 3-kinase inhibitor LY294002 obviated the ability to sense substrate stiffness, suggesting that phosphatidylinositol 3-kinase plays a mechanistic role in neutrophil mechanosensing.

Macrophage phagocytosis of wound neutrophils.

Resolution of acute inflammation is thought to require the recognition and phagocytosis of apoptotic neutrophils (PMN) through receptor‐ligand interactions with macrophages (Mϕ). This hypothesis was tested in rat wounds by quantifying apoptosis in freshly harvested and aged‐in‐culture PMN taken from wounds 1–3 days after injury and by using these wound PMN as phagocytic targets for wound, immune‐activated peritoneal, and resident peritoneal Mϕ. Less than 6% of freshly harvested PMN exhibited characteristics of apoptosis. On aging in culture, day 1 PMN did not undergo apoptosis, whereas 41 ± 1 and 29 ± 1% of day 2 and 3 PMN, respectively, developed apoptosis, which corresponded to increased ingestion by Mϕ. All three Mϕ populations engaged different receptor‐ligand pairs for the recognition and phagocytosis of PMN. Results indicate the resistance of early wound PMN to age‐induced apoptosis, demonstrate wound‐Mϕ phagocytosis of wound PMN, and identify distinct receptor utilization by wound and other Mϕ to ingest wound PMN. J. Leukoc. Biol. 65:35–42; 1999.

Molecular and Metabolic Evidence for the Restricted Expression of Inducible Nitric Oxide Synthase in Healing Wounds

Tissue injury initiates a temporally ordered sequence of local cellular and metabolic responses presumably necessary for successful repair. Previous investigations demonstrated that metabolic evidence for nitric oxide synthase (NOS) activity is detectable in wounds only during the initial 48 to 72 hours of the repair process. Present results identify the cell types contributing inducible NOS (iNOS) to experimental wounds in rats. iNOS antigen was expressed in most macrophages present in wounds 6 to 24 hours after injury, and these cells exhibited NAPDH diaphorase and NOS activity. Polymorphonuclear leukocytes contained little iNOS antigen and no NADPH diaphorase activity and were minimally able to convert L-arginine to L-citrulline. The frequency of iNOS-positive macrophages declined on days 3 and 5 after wounding. By day 10, most macrophages in the wound were negative for iNOS. These cells, however, acquired iNOS antigen and activity in culture. Wound fluids, but not normal rat serum, suppressed the induction of iNOS during culture. Findings indicate that the expression of iNOS in healing wounds is restricted to macrophages present during the early phases of repair and that components of wound fluid suppress the induction of iNOS in macrophages in late wounds. Polymorphonuclear leukocytes contribute little iNOS activity to the healing wound.

Recombinant human activated protein C inhibits integrin-mediated neutrophil migration

Integrin-mediated cell migration is central to many biologic and pathologic processes. During inflammation, tissue injury results from excessive infiltration and sequestration of activated leukocytes. Recombinant human activated protein C (rhAPC) has been shown to protect patients with severe sepsis, although the mechanism underlying this protective effect remains unclear. Here, we show that rhAPC directly binds to beta(1) and beta(3) integrins and inhibits neutrophil migration, both in vitro and in vivo. We found that human APC possesses an Arg-Gly-Asp (RGD) sequence, which is critical for the inhibition. Mutation of this sequence abolished both integrin binding and inhibition of neutrophil migration. In addition, treatment of septic mice with a RGD peptide recapitulated the beneficial effects of rhAPC on survival. Thus, we conclude that leukocyte integrins are novel cellular receptors for rhAPC and the interaction decreases neutrophil recruitment into tissues, providing a potential mechanism by which rhAPC may protect against sepsis.