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We previously showed that anti–neutrophil extracellular trap (NET) rheumatoid arthritis (RA)-rmAbs derived from CD19+ B cells within RA human synovial tissues frequently react against NETs. In this study, we aimed to characterize the importance of affinity maturation via somatic hypermutation (SHM) within the Ig variable H (VH) and variable L (VL) chains and Fab–N-linked glycosylation in RA synovial B cell clones reactive to NETs and NET-derived Ags such as citrullinated histones. Selected anti-NET RA-rmAbs derived from synovial RA CD19+ B cells were subjected to overlap-PCR to generate germline (GL; VH and VL reverted into GL), hybrid clones (VH/VL region reverted into GL), and N-glycosylation mutants (N→Q) and analyzed for anti-NETs and citrullinated histones (cit-H2B) immunoreactivity. Anti-NET/cit-H2B immunoreactivity of selected RA-rmAbs was abrogated in the VH and VL GL counterpart. In RA B cell hybrid clone RA015/11.88 and RA056/11.23.2, NET and/or cit-H2B immunoreactivity was solely dependent on SHM in the IgVH region whereas RA B cell hybrid clone RA015/11.91 required affinity maturation of both VH and VL for efficient binding to cit-H2B. In 7/80 RA-rmAb, SHM resulted in ex novo N-glycosylation sites in VH and/or VL regions. Removal of Fab-linked glycans in RA056/11.23.2 in the N-mutant counterpart resulted in 90% reduction in immunoreactivity to cit-H2B. Thus, SHM in the IgVH and/or VL regions of RA synovial B cells is necessary for the immunoreactivity to NET-Ags. Fab–N-linked–glycosylation introduction sites are observed in a minority of anti-NET B cell clones but can strongly influence NET-Ag binding.

We hypothesized that exudates collected at the beginning of the resolution phase of inflammation might be enriched for tissue protective molecules; thus an integrated cellular and molecular approach was applied to identify novel chondroprotective bioactions. Exudates were collected 6 h (inflammatory) and 24 h (resolving) following carrageenan-induced pleurisy in rats. The resolving exudate was subjected to gel filtration chromatography followed by proteomics, identifying 61 proteins. Fractions were added to C28/I2 chondrocytes, grown in micromasses, ions with or without IL-1β or osteoarthritic synovial fluids for 48 h. Three proteins were selected from the proteomic analysis, α1-antitrypsin (AAT), hemopexin (HX), and gelsolin (GSN), and tested against catabolic stimulation for their effects on glycosaminoglycan deposition as assessed by Alcian blue staining, and gene expression of key anabolic proteins by real-time PCR. In an in vivo model of inflammatory arthritis, cartilage integrity was determined histologically 48 h after intra-articular injection of AAT or GSN. The resolving exudate displayed protective activities on chondrocytes, using multiple readouts: these effects were retained in low m.w. fractions of the exudate (46.7% increase in glycosaminoglycan deposition; ∼20% upregulation of COL2A1 and aggrecan mRNA expression), which reversed the effect of IL-1β. Exogenous administration of HX, GSN, or AAT abrogated the effects of IL-1β and osteoarthritic synovial fluids on anabolic gene expression and increased glycosaminoglycan deposition. Intra-articular injection of AAT or GSN protected cartilage integrity in mice with inflammatory arthritis. In summary, the strategy for identification of novel chondroprotective activities in resolving exudates identified HX, GSN and AAT as potential leads for new drug discovery programs.

There is a need for novel approaches to control pathologies with overexuberant inflammatory reactions. Targeting melanocortin (MC) receptors represents a promising therapy for obesity and chronic inflammation, but lack of selectivity and safety concerns limit development. A new way to increase selectivity of biological effects entails the identification of biased agonists. In this study, we characterize the small molecule AP1189 as a biased agonist at receptors MC1 and MC3. Although not provoking canonical cAMP generation, AP1189 addition to MC1 or MC3, but not empty vector, transfected HEK293 cells caused ERK1/2 phosphorylation, a signaling responsible for the proefferocytic effect evoked in mouse primary macrophages. Added to macrophage cultures, AP1189 reduced cytokine release, an effect reliant on both MC1 and MC3 as evident from the use of Mc1r−/− and Mc3r−/− macrophages. No melanogenesis was induced by AP1189 in B16-F10 melanocytes. In vivo, oral AP1189 elicited anti-inflammatory actions in peritonitis and, upon administration at the peak of inflammation, accelerated the resolution phase by ∼3-fold. Finally, given the clinical efficacy of adrenocorticotropin in joint diseases, AP1189 was tested in experimental inflammatory arthritis, where this biased agonist afforded significant reduction of macroscopic and histological parameters of joint disruption. These proof-of-concept analyses with AP1189, an active oral anti-inflammatory and resolution-promoting compound, indicate that biased agonism at MC receptors is an innovative, viable approach to yield novel anti-inflammatory molecules endowed with a more favorable safety profile.

Little is known about the role(s) of endogenous galectin-1 (Gal-1) in arthritis. In this study we queried whether antiarthritic functions for this effector of endogenous anti-inflammation could be unveiled by studying collagen-induced arthritis in Gal-1−/− mice. Gal-1−/− and C57BL/6J [wild-type (WT)] mice received an immunization of chicken type II collagen (CII) in CFA followed by a booster on day 21, which consisted of CII in IFA. Animals were monitored for signs of arthritis from day 14 onward. Clinical and histological signs of arthritis were recorded, and humoral and cellular immune responses against CII were analyzed. A distinct disease penetrance was apparent, with ∼ 70% of Gal-1−/− mice developing arthritis compared with ∼ 50% in WT animals. Gal-1−/− mice also exhibited an accelerated disease onset and more severe arthritis characterized by significantly elevated clinical scores. Postmortem analyses (day 42) revealed higher levels of IgG1 and IgG2b anti-CII Ig isotypes in the serum of Gal-1 null animals compared with WT. Finally, T cell responses following ex vivo stimulation with CII revealed a greater degree of proliferation in T cells of Gal-1−/− mice compared with WT, which was associated with increased production of IL-17 and IL-22. These data suggest the novel idea that endogenous Gal-1 is an inhibitory factor in the development of arthritis affecting disease severity. We have also highlighted the importance of endogenous Gal-1 in regulating T cell reactivity during experimental arthritis.

Endogenous microparticles (MPs) were systematically profiled during the time course of self-limited inflammation. Precursors for specialized proresolving lipid mediators were identified in MPs from inflammatory exudates using liquid chromatography tandem mass spectrometry-based metabolomics. Hence, we postulated that formation of anti-inflammatory and proresolving lipid mediators could underlie beneficial effects attributed to MPs and that this process could serve as a basis for biomimicry. Using human neutrophil-derived MPs, we constructed novel nanoparticles (NPs) containing aspirin-triggered resolvin D1 or a lipoxin A4 analog. Enriched NPs dramatically reduced polymorphonuclear cell influx in murine peritonitis, shortened resolution intervals, and exhibited proresolving actions accelerating keratinocyte healing. The enriched NPs protected against inflammation in the temporomandibular joint. These findings indicate that humanized NPs, termed nano-proresolving medicines, are mimetics of endogenous resolving mechanisms, possess potent beneficial bioactions, can reduce nanotoxicity, and offer new therapeutic approaches.

The importance of proresolving mediators in the overall context of the resolution of acute inflammation is well recognized, although little is known about whether these anti-inflammatory and proresolving molecules act in concert. In this article, we focused on lipoxin A4 (LXA4) and annexin A1 (AnxA1) because these two very different mediators converge on a single receptor, formyl peptide receptor type 2 (FPR2/ALX). Addition of LXA4 to human polymorphonuclear leukocytes (PMNs) provoked a concentration- and time-dependent mobilization of AnxA1 onto the plasma membrane, as determined by Western blotting and flow cytometry analyses. This property was shared by another FPR2/ALX agonist, antiflammin-2, and partly by fMLF or peptide Ac2-26 (an AnxA1 derivative that can activate all three members of the human FPR family). An FPR2/ALX antagonist blocked AnxA1 mobilization activated by LXA4 and antiflammin-2. Analysis of PMN degranulation patterns and phospho-AnxA1 status suggested a model in which the two FPR2/ALX agonists mobilize the cytosolic (and not the granular) pool of AnxA1 through an intermediate phosphorylation step. Intravital microscopy investigations of the inflamed mesenteric microvasculature of wild-type and AnxA1−/− mice revealed that LXA4 provoked leukocyte detachment from the postcapillary venule endothelium in the former (>50% within 10 min; p < 0.05), but not the latter genotype (∼15%; NS). Furthermore, recruitment of Gr1+ cells into dorsal air-pouches, inflamed with IL-1β, was significantly attenuated by LXA4 in wild-type, but not AnxA1−/−, mice. Collectively, these data prompt us to propose the existence of an endogenous network in anti-inflammation centered on PMN AnxA1 and activated by selective FPR2/ALX agonists.

The human formyl-peptide receptor (FPR)-2 is a G protein-coupled receptor that transduces signals from lipoxin A4, annexin A1, and serum amyloid A (SAA) to regulate inflammation. In this study, we report the creation of a novel mouse colony in which the murine FprL1 FPR2 homologue, Fpr2, has been deleted and describe its use to explore the biology of this receptor. Deletion of murine fpr2 was verified by Southern blot analysis and PCR, and the functional absence of the G protein-coupled receptor was confirmed by radioligand binding assays. In vitro, Fpr2−/− macrophages had a diminished response to formyl-Met-Leu-Phe itself and did not respond to SAA-induced chemotaxis. ERK phosphorylation triggered by SAA was unchanged, but that induced by the annexin A1-derived peptide Ac2–26 or other Fpr2 ligands, such as W-peptide and compound 43, was attenuated markedly. In vivo, the antimigratory properties of compound 43, lipoxin A4, annexin A1, and dexamethasone were reduced notably in Fpr2−/− mice compared with those in wild-type littermates. In contrast, SAA stimulated neutrophil recruitment, but the promigratory effect was lost following Fpr2 deletion. Inflammation was more marked in Fpr2−/− mice, with a pronounced increase in cell adherence and emigration in the mesenteric microcirculation after an ischemia–reperfusion insult and an augmented acute response to carrageenan-induced paw edema, compared with that in wild-type controls. Finally, Fpr2−/− mice exhibited higher sensitivity to arthrogenic serum and were completely unable to resolve this chronic pathology. We conclude that Fpr2 is an anti-inflammatory receptor that serves varied regulatory functions during the host defense response. These data support the development of Fpr2 agonists as novel anti-inflammatory therapeutics.

During resolution of an inflammatory response, recruited neutrophil granulocytes undergo apoptosis and are removed by tissue phagocytes before induction of secondary necrosis without provoking proinflammatory cytokine production and release. Promotion of physiological neutrophil clearance mechanisms may represent a viable therapeutic strategy for the treatment of inflammatory or autoimmune diseases in which removal of apoptotic cells is impaired. The mechanism underlying enhancement of macrophage capacity for phagocytosis of apoptotic cells by the powerful anti-inflammatory drugs of the glucocorticoid family has remained elusive. In this study, we report that human monocyte-derived macrophages cultured in the presence of dexamethasone exhibit augmented capacity for phagocytosis of membrane-intact, early apoptotic cells only in the presence of a serum factor. Our results eliminate a role for a number of potential opsonins, including complement, pentraxin-3, and fibronectin. Using ion-exchange and gel filtration chromatography, we identified a high molecular mass serum fraction containing C4-binding protein and protein S responsible for the augmentation of phagocytosis of apoptotic neutrophils. Because the apoptotic neutrophils used in this study specifically bind protein S, we suggest that glucocorticoid treatment of macrophages induces a switch to a protein S-dependent apoptotic cell recognition mechanism. Consistent with this suggestion, pretreatment of macrophages with Abs to Mer tyrosine kinase, a member of the Tyro3/Axl/Mer family of receptor tyrosine kinases, prevented glucocorticoid augmentation of phagocytosis. Induction of a protein S/Mer tyrosine kinase-dependent apoptotic cell clearance pathway may contribute to the potent anti-inflammatory effects of glucocorticoids, representing a potential target for promoting resolution of inflammatory responses.

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (−/−) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (−/−) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.

Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE−/−) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE−/− mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1β and TNF-α and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE−/− mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1β, KC, and macrophage inflammatory protein-1α in NE−/− mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.

The issue of which melanocortin receptor (MC-R) is responsible for the anti-inflammatory effects of melanocortin peptides is still a matter of debate. Here we have addressed this aspect using a dual pharmacological and genetic approach, taking advantage of the recent characterization of more selective agonists/antagonists at MC1 and MC3-R as well as of the existence of a naturally defective MC1-R mouse strain, the recessive yellow (e/e) mouse. RT-PCR and ultrastructural analyses showed the presence of MC3-R mRNA and protein in peritoneal macrophages (Mφ) collected from recessive yellow (e/e) mice and wild-type mice. This receptor was functional as Mφ incubation (30 min) with melanocortin peptides led to accumulation of cAMP, an effect abrogated by the MC3/4-R antagonist SHU9119, but not by the selective MC4-R antagonist HS024. In vitro Mφ activation, determined as release of the CXC chemokine KC and IL-1β, was inhibited by the more selective MC3-R agonist γ2-melanocyte stimulating hormone but not by the selective MC1-R agonist MS05. Systemic treatment of mice with a panel of melanocortin peptides inhibited IL-1β release and PMN accumulation elicited by urate crystals in the murine peritoneal cavity. MS05 failed to inhibit any of the inflammatory parameters either in wild-type or recessive yellow (e/e) mice. SHU9119 prevented the inhibitory actions of γ2-melanocyte stimulating hormone both in vitro and in vivo while HS024 was inactive in vivo. In conclusion, agonism at MC3-R expressed on peritoneal Mφ leads to inhibition of experimental nonimmune peritonitis in both wild-type and recessive yellow (e/e) mice.

The effect of the glucocorticoid inducible protein annexin 1 (ANXA1) on the process of monocytic cell migration was studied using transfected U937 cells expressing variable protein levels. An antisense (AS) (36.4AS; ∼50% less ANXA1) and a sense (S) clone (15S; overexpressing the bioactive 24-kDa fragment) together with the empty plasmid CMV clone were obtained and compared with wild-type U937 cells in various models of cell migration in vitro and in vivo. 15S-transfected U937 cells displayed a reduced (50%) degree of trans-endothelial migration in response to stromal cell-derived factor-1α (CXC chemokine ligand 12 (CXCL12)). In addition, the inhibitory role of endogenous ANXA1 on U937 cell migration in vitro was confirmed by the potentiating effect of a neutralizing anti-ANXA1 serum. Importantly, overexpression of ANXA1 in clone 15S inhibited the extent of cell migration into rheumatoid synovial grafts transplanted into SCID mice. ANXA1 inhibitory effects were not due to modifications in adhesion molecule or CXCL12 receptor (CXCR4) expression as shown by the similar amounts of surface molecules found in transfected and wild-type U937 cells. Likewise, an equal chemotactic response to CXCL12 in vitro excluded an intrinsic defect in cell motility in clones 15S and 36.4AS. These data strongly support the notion that ANXA1 critically interferes with a leukocyte endothelial step essential for U937 cell, and possibly monocyte, transmigration both in vitro and in vivo.

Stromal cell-derived factor 1 (SDF-1; CXCL12), a CXC chemokine, has a primary role in signaling the recruitment of hemopoietic stem cell precursors to the bone marrow during embryonic development. In postnatal life, SDF-1 is widely expressed and is induced in chronically inflamed tissues such as psoriatic skin and the rheumatoid synovium, but has also been implicated in the migration of lymphocytes to lymphoid organs. To investigate the role of SDF-1 in recirculation and homing in vivo, we have developed a model in which human peripheral lymph nodes (huPLN) are transplanted into SCID mice. We have shown that huPLN transplants are viable, vascularized by the murine circulation that forms functional anastomoses with transplant vessels. In addition, grafts retain some features of the pretransplantation tissue, such as lymphoid follicles, lymphatic and high endothelial venule markers. We also show that SDF-1 is capable of inducing the migration of a SDF-1-responsive cell line (U937) and human PBLs from the murine circulation into the grafts in a dose-dependant manner, inhibitable by CXCR4 blockade. The mechanism of action of SDF-1 in this model is independent from that of TNF-α and does not rely on the up-regulation of adhesion molecules (such as ICAM-1) on the graft vascular endothelium. This is the first description of huPLN transplantation into SCID mice and of the functional effects of SDF-1 in regard to the migration of human cells into huPLN in vivo. This model provides a powerful tool to investigate the pathways involved in cell migration into lymphoid organs and potentially to target them for therapeutic purposes.

To investigate the relevance of adrenocorticotrophic hormone (ACTH) therapy in human gouty arthritis, we have tested the effect of several ACTH-related peptides in a murine model of experimental gout. Systemic treatment of mice with ACTH4–10 (MEHFRWG) (10–200 μg s.c.) inhibited neutrophil accumulation without altering peripheral blood cell counts or circulating corticosterone levels. A similar effect was seen with α- and β-melanocyte stimulating hormones (1–30 μg s.c.). In vivo release of the chemokine KC-(detected in the lavage fluids before maximal influx of neutrophils) was significantly reduced (−50 to −60%) by ACTH4–10. Macrophage activation in vitro, determined as phagocytosis and KC release, was inhibited by ACTH and ACTH4–10 with approximate IC50 values of 30 nM and 100 μM, respectively. The melanocortin receptor type 3/4 antagonist SHU9119 prevented the inhibitory actions of ACTH4–10 both in vitro and in vivo. However, melanocortin type 3, but not type 4, receptor mRNA was detected in mouse peritoneal macrophages by RT-PCR. Therefore, we propose that activation of this receptor type by ACTH4–10 and related amino acid sequences attenuates KC release (and possibly production of other cytokines) from macrophages with consequent inhibition of the host inflammatory response, thus providing a notional anti-inflammatory mechanism for ACTH that is unrelated to stimulation of glucocorticoid release.

The roles played by resident macrophages (Mφ) and mast cells (MCs) in polymorphonuclear leukocyte (PMN) accumulation and chemokine production within the mouse peritoneal cavity in response to administration of zymosan (0.2 and 1 mg), LPS (1 mg/kg), and thioglycolate (0.5 ml of a 3% suspension) were investigated. A marked reduction (>95%) in intact MC numbers was obtained by pretreatment with the MC activator compound 48/80, whereas resident Mφ were greatly diminished (>85%) by a 3-day treatment with liposomes encapsulating the cytotoxic drug dichloromethylene-bisphosphonate. No modulation of thioglycolate-induced inflammation was seen with either pretreatment. Removal of either MCs or Mφ attenuated LPS-induced PMN extravasation without affecting the levels of the chemokines murine monocyte chemoattractant protein-1 and KC measured in the lavage fluids. In contrast, MC depletion inhibited PMN accumulation and murine monocyte chemoattractant protein-1 and KC production in the zymosan peritonitis model. Removal of Mφ augmented the accumulation of PMN elicited by the latter stimulus. This was due to an inhibitory action of Mφ-derived IL-10 because there was 1) a time-dependent release of IL-10 in the zymosan exudates; 2) a reduction in IL-10 levels following Mφ, but not MC, depletion; and 3) an increased PMN influx and chemokine production in IL-10 knockout mice. In conclusion, we propose a stimulus-dependent role of resident MCs in chemokine production and the existence of a regulatory loop between endogenous IL-10 and the chemokine-mediated cellular component of acute inflammation.

Inflammation-induced epithelial injury results in compromised mucosal barrier function. In response to injury, epithelial cells migrate as a sheet to cover denuded surfaces. We have recently reported that a calcium dependent phospholipid binding protein Annexin A1 (AnxA1) is a pro-resolving mediator that signals via epithelial formyl peptide receptors (FPRs) to promote intestinal mucosal wound repair. Analysis of signaling pathways downstream of epithelial FPRs identified intestinal epithelial NADPH oxidase-1 (Nox1) and Rac1 as key elements responsible for generation of reactive oxygen species (ROS). We determined that ROS-induced oxidative modification of phosphatases, in turn, activate proteins involved in controlling focal cell matrix adhesions and epithelial cell migration. Exogenous AnxA1 administration was observed to promote intestinal mucosal wound closure in WT mice but not in mice lacking intestinal epithelial Nox1 (Nox1-/-IEC). Analysis of AnxA1 released from the epithelial cells revealed that it is shed in membranes that have properties of microparticles (