Jorge E. Albina

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.

Wound Macrophages as Key Regulators of Repair: Origin, Phenotype, and Function

Recent results call for the reexamination of the phenotype of wound macrophages and their role in tissue repair. These results include the characterization of distinct circulating monocyte populations with temporally restricted capacities to migrate into wounds and the observation that the phenotype of macrophages isolated from murine wounds partially reflects those of their precursor monocytes, changes with time, and does not conform to current macrophage classifications. Moreover, findings in genetically modified mice lacking macrophages have confirmed that these cells are essential to normal wound healing because their depletion results in retarded and abnormal repair. This mini-review focuses on current knowledge of the phenotype of wound macrophages, their origin and fate, and the specific macrophage functions that underlie their reparative role in injured tissues, including the regulation of the cellular infiltration of the wound and the production of transforming growth factor-β and vascular endothelial growth factor.

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.

On the expression of nitric oxide synthase by human macrophages. Why no NO

The production of nitric oxide (NO) and its role in the anti‐tumor and anti‐microbial effects of rodent macrophages appears well established. In contrast, the circumstances required for its release from human monocytes/macrophages and its potential role in human pathology remain controversial. Evidence to be discussed suggests that NO is a redundant, autotoxic, immunosuppressive, and inefficient mediator of macrophage function. For these reasons, the expression of nitric oxide synthase as a rapid‐response, high‐output effector pathway may have been evolved out of the human monocyte/macrophage response repertoire or severely restricted in its expression. Hypothetical roles for a modest and circumscribed production of NO by human macrophages are proposed.

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.

Suppression of lymphocyte proliferation through the nitric oxide synthesizing pathway

The amino acid L-arginine can be metabolized through a nitric oxide-synthesizing pathway (NOSP) to produce L-citrulline and reactive nitrogen intermediates. Among these nitrogen intermediates, NO has been implicated as the mediator of a variety of biological effects including vasodilatation, inhibition of platelet aggregation, tumor cytotoxicity and microbiostasis by activated macrophages and generalized suppression of macrophage functions. Work reported here demonstrated that the NOSP is expressed in Con A-stimulated rat splenic cell (SC) cultures and is associated with a profound suppression of lymphocyte proliferation. Inhibition of the NOSP by NG-monomethyl-L-arginine (N-MMA) or binding of its products by hemoglobin, either free in solution or contained in RBC, markedly promotes rat SC mitogenic response to Con A. Mouse SC do not express the NOSP under the conditions used in these experiments. Consequently, their mitogenic response to Con A is not affected by N-MMA or hemoglobin. These data confirm and expand the apparent role of NO as a regulator of immune responses while indicating potentially important species differences.

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.

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.

Nutrition and wound healing.

A role for nutrition therapy in the regulation of wound healing has been predicated on two concepts. The first is that malnutrition increases the risk of wound-related complications. The second is that dietary intervention, either in the form of complete nutrition support or as single nutrient supplementation, can improve or accelerate the wound-healing response. Although frequently stated and extensively discussed, these two concepts are not supported by objective data.

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.