The CD36 and SR-A/CD204 scavenger receptors fine-tune Staphylococcus aureus-stimulated cytokine production in mouse macrophages.

The occurring in SR-A/CD204- or CD36-deficient mice increased susceptibility to infections with Staphylococcus aureus (Sa) had traditionally been ascribed to the impairment of macrophage-mediated phagocytosis, which is, however, inconsistent with low effectiveness of unopsonized Sa killing within macrophages and redundant roles of both receptors in this process. We have found that Sa-stimulated cytokine production in mouse macrophages seems to be exclusively mediated by TLR2, mainly from within endosomes in response to Sa-derived lipoteichoic acid. By driving endocytic trafficking of TLR2 and its ligands through the clathrin-dependent pathway, CD36 and SR-A sensitize macrophages to activation by Sa as well as regulate the type and amount of cytokines produced. Additionally, upon direct Sa binding, both receptors autonomously generate anti-inflammatory signaling. Consequently, the delayed induction of acute inflammation in knockout mice may allow for the initial, uncontrolled multiplication of bacteria, stimulating excessive, septic shock-inducing production of inflammatory cytokines in later stages of infection.

Phagocytosis of live versus killed or fluorescently labeled bacteria by macrophages differ in both magnitude and receptor specificity.

Scavenger receptor (SR)-mediated opsonin-independent phagocytosis of bacteria by macrophages has been suggested to represent an important, early mechanism of anti-bacterial host defense. However, although the ability to bind bacteria has been demonstrated to be a shared feature of all types of SRs, in many cases the evidence is limited to the demonstration of increased binding of killed, fluorescently labeled bacteria to non-phagocytic cells transfected with these receptors. We sought to verify the ability of SRs to mediate non-opsonic phagocytosis of live Escherichia coli (Ec) and Staphylococcus aureus (Sa), model species of Gram-negative and -positive bacteria, respectively, and to assess the relative contributions of different SRs expressed on murine macrophages in this process. We found that the class A SR SR-A/CD204 was the major receptor mediating phagocytosis of fluorescently labeled Sa, whereas different SRs had highly redundant roles in the phagocytosis of live Sa. Conversely, different SRs contributed to the phagocytosis of fluorescently labeled Ec. In comparison, phagocytosis of live Ec was of much lower magnitude and was selectively mediated by SR-A. These results question the use of fluorescently labeled bacteria as valid replacements for live bacteria. The low magnitude of opsonin-independent phagocytosis of Ec and unimpaired phagocytosis of Sa in SR-A- or CD36-deficient macrophages indicate that the defect in this process might not be responsible for the reported impaired bacteria clearance in mice deficient in these receptors. We postulate that this impairment might result to a larger extent from inhibition of intracellular bacteria killing caused by pro-inflammatory cytokines, produced in excessive amounts by SR-deficient cells in response to bacterial products.

CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide.

Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern of Gram-negative bacterial infections, and includes smooth (S-LPS) and rough (R-LPS) chemotypes. Upon activation by LPS through CD14, TLR4/MD-2 heterodimers sequentially induce two waves of intracellular signaling for macrophage activation: the MyD88-dependent pathway from the plasma membrane and, following internalization, the TRIF-dependent pathway from endosomes. We sought to better define the role of scavenger receptors CD36 and CD204/SR-A as accessory LPS receptors that can contribute to pro-inflammatory and microbicidal activation of macrophages. We have found that CD36 differently regulates activation of mouse macrophages by S-LPS versus R-LPS. The ability of CD36 to substitute for CD14 in loading R-LPS, but not S-LPS onto TLR4/MD-2 allows CD14-independent macrophage responses to R-LPS. Conversely, S-LPS, but not R-LPS effectively stimulates CD14 binding to CD36, which favors S-LPS transfer from CD14 onto TLR4/MD-2 under conditions of low CD14 occupancy with S-LPS in serum-free medium. In contrast, in the presence of serum, CD36 reduces S-LPS binding to TLR4/MD-2 and the subsequent MyD88-dependent signaling, by mediating internalization of S-LPS/CD14 complexes. Additionally, CD36 positively regulates activation of TRIF-dependent signaling by both S-LPS and R-LPS, by promoting TLR4/MD-2 endocytosis. In contrast, we have found that SR-A does not function as a S-LPS receptor. Thus, by co-operating with CD14 in both R- and S-LPS loading onto TLR4/MD-2, CD36 can enhance the sensitivity of tissue-resident macrophages in detecting infections by Gram-negative bacteria. However, in later phases, following influx of serum to the infection site, the CD36-mediated negative regulation of MyD88-dependent branch of S-LPS-induced TLR4 signaling might constitute a mechanism to prevent an excessive inflammatory response, while preserving the adjuvant effect of S-LPS for adaptive immunity.

Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect infections caused by pathogens not recognized by pattern recognition receptors.

The class A scavenger receptor SR-A/CD204 and the class B scavenger receptor CD36 regulate immune functions of macrophages differently.

SR-A/CD204 and CD36 are major receptors responsible for oxidized lipoproteins uptake by macrophages in atherosclerotic plaques. Both receptors also share the role as receptors for different pathogens, but studies on their signaling have been hampered by the lack of selective ligands. We report that, upon specific ligation by Ab, SR-A does not induce cytokine production, but mediates inhibition of LPS-stimulated production of IL-6 and IL-12/23p40, enhancement of IL-10 release, and has no effect on TNF-α and RANTES production in murine macrophages. In contrast, anti-CD36 Ab alone stimulated production of all these cytokines, with IL-10 production being exceptionally high. Effects of anti-CD36 Ab, except of IL-10 production, were mediated by CD14 and TLR2, whereas those of SR-A ligation by heterotrimeric Gi/o proteins and by phosphatidylinositol 3-kinases. Surprisingly, we found that LPS uptake by macrophages was mediated in part by CD36 cooperating with CD14, whereas SR-A was not involved in this process. Finely, during in vitro Ag presentation to naïve CD4(+) lymphocytes, pre-incubation of macrophages with anti-CD36 Ab enhanced IFN-γ production in the co-culture, but exerted the opposite effect under conditions enabling IL-10 accumulation. In contrast, anti-SR-A Ab was ineffective alone, but reversed the Th1-polarizing effect of LPS.

Stiffness changes of tumor HEp2 cells correlates with the inhibition and release of TRAIL-induced apoptosis pathways.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) is a promising apoptotic agent that can selectively act on tumor cells. However, some cancer cells are resistant to TRAIL mediated apoptosis. In specific type of cells, sensitization by chemotherapeutic drugs may overcome the resistance to TRAIL induced apoptosis. In this work, atomic force microscopy (AFM) nanoindentation spectroscopy combined with fluorescence methods were used to investigate the biomechanical aspects of the resistance and unblocking of apoptosis in larynx carcinoma HEp2 cells treated with TRAIL. It is shown that there is a direct correlation between the increase in mechanical cell stiffness and the inhibition of apoptosis induced by TRAIL in HEp2 cells. Conversely, unblocking of apoptosis by sensitization of HEp2 cells with a chemotherapeutic drug Actinomycin D is related to the depolymerization of F-actin and to the decrease in the cell stiffness. Both effects, that is, changes in the mechanical stiffness of the cell and the inhibition of apoptotic pathway, are closely related to the Bcl-2 activity. Most probably, the depolymerization of F-actin results from downregulation of Rho protein, which in turn is accompanied by a lower activity of Bcl-2 and in consequence releases the intrinsic apoptotic channel. The presented results reveal a promising application of nanoindentation spectroscopy with an AFM tip as a novel tool for monitoring the processes of apoptosis inhibition.

Atomic force microscopy-based molecular studies on the recognition of immunogenic chlorinated ovalbumin by macrophage receptors.

This report presents simple and reliable approach developed to study the specific recognition events between chlorinated ovalbumin (OVA) and macrophages using atomic force microscopy (AFM). Thanks to the elimination of nonspecific adhesion, the interactions of the native and chlorinated OVA with a membrane of macrophages could be quantified using exclusively the so-called adhesion frequency (AF). The proposed system not only enabled the application of AFM-based force measurements for such poorly defined ligand-receptor pairs but also significantly improved both the acquisition and the processing of the data. The proteins were immobilized on the gold-coated AFM tips from the aqueous solutions containing charged thiol adsorbates. Such surface dilution of the proteins ensured the presence of single or just a few macromolecules at the tip-surface contact. The formation of negatively charged monolayer on the tip dramatically limited its nonspecific interactions with the macrophage surface. In such systems, AF was used as a measure of the recognition events even if the interaction forces varied significantly for sets of measurements. The system with the native OVA, a weak immunogen, showed only negligible AF compared with 85% measured for the immunogenic chlorinated OVA. The AF values varied with the tip-macrophage contact time and loading velocity. Blocking of the receptors by the chlorinated OVA was also confirmed. The developed approach can be also used to study other ligand-receptor interactions in poorly defined biological systems with intrinsically broad distribution of the rupture forces, thus opening new fields for AFM-based recognition on molecular level.

Hypochlorous acid: a natural adjuvant that facilitates antigen processing, cross-priming, and the induction of adaptive immunity.

The production of hypochlorous acid (HOCl) is a characteristic of granulocyte activation, a hallmark of the early phase of innate immune responses. In this study, we show that, in addition to its well-established role as a microbicide, HOCl can act as a natural adjuvant of adaptive immunity. HOCl enhances the T cell responses to the model Ag OVA, facilitating the processing and presentation of this protein via the class II MHC pathway. HOCl modification also enhances cross-presentation of the tumor Ag tyrosinase-related protein 2 via class I MHC. The adjuvant effects of HOCl are independent of TLR signaling. The enhanced presentation of HOCl-modified OVA is mediated via modification of the N-linked carbohydrate side chain rather than formation of protein aldehydes or chloramines. HOCl-modified OVA is taken up more efficiently by APCs and is degraded more efficiently by proteinases. Atomic force microscopy demonstrated that enhanced uptake is mediated via specific receptor binding, one candidate for which is the scavenger receptor lectin-like oxidized low-density lipoprotein receptor, which shows enhanced binding to chlorinated OVA. A function of HOCl is therefore to target glycoprotein Ags to scavenger receptors on the APC surface. This additional mechanism linking innate and adaptive immunity suggests novel strategies to enhance immunity to vaccines.

Taurine haloamines and heme oxygenase-1 cooperate in the regulation of inflammation and attenuation of oxidative stress.

Taurine chloramine (TauCl) and Taurine bromamine (TauBr), products of the neutrophil myeloperoxidase halide system, exert anti-inflammatory properties. They inhibit the production of a variety of inflammatory mediators, such as prostaglandin E2 (PGE2), nitric oxide (NO) and proinflammatory cytokines. Heme oxygenase-1 (HO-1), a stress inducible enzyme, degrades heme to biliverdin, free iron and carbon monoxide (CO), which are involved in the anti-inflammatory and antioxidant actions of HO-1. Recently we have demonstrated that taurine haloamines induce the expression of HO-1 in inflammatory cells. In this study we examined whether HO-1 participates in taurine haloamines-mediated suppression of proinflammatory cytokine production. We have shown that TauCl/TauBr and CO inhibit the production of TNF-alpha, IL-12 and IL-6, in a similar dose-dependent manner. However, the suppressor activity of TauCl was not altered in HO-1 deficient mice. Therefore, HO-1 and TauCl may independently regulate the production of proinflammatory cytokines. We suggest that TauCl and TauBr provide a link between the two antioxidant systems: the cysteine pathway and the heme oxygenase system.

1-Methylnicotinamide and nicotinamide: two related anti-inflammatory agents that differentially affect the functions of activated macrophages.

INTRODUCTION: 1-Methylnicotinamide (MNA), a major metabolite of nicotinamide (NA), is known to exert anti-inflammatory effects in vivo. Treatment of inflammatory skin diseases by topical application of MNA provides certain advantages over the use of NA. However, in contrast to NA, the molecular mechanisms of the anti-inflammatory properties of MNA are not well known. In this study the influence of exogenous MNA and NA in vivo on the generation of inflammatory mediators by macrophages (Mvarphi) was investigated. MATERIALS AND METHODS: Peritoneal Mvarphi of CBA/J mice were activated in vitro with lipopolysaccharide and incubated with MNA or NA. The effect of these compounds on biological functions of Mvarphi was measured by evaluation of the production of reactive oxygen species (ROS) by luminol-dependent chemiluminescence, cytokines and prostaglandin E(2) (PGE(2)) by ELISA, and nitric oxide (NO) by the Griess method. Moreover, the expressions of inducible NO synthase and cyclooxygenase-2 were measured by Western blotting. RESULTS: It was shown that at non-cytotoxic concentrations, NA inhibits the production of a variety of pro-inflammatory agents, such as tumor necrosis factor alpha, interleukin 6, NO, PGE(2), and the generation of ROS. In contrast to NA, exogenous MNA inhibited only the generation of ROS, while its effect on the synthesis of other mediators was negligible. CONCLUSIONS: These results indicate that the anti-inflammatory properties of MNA demonstrated previously in vivo do not depend on its capacity to suppress the functions of immune cells, but more likely may be related to its action on vascular endothelium. The authors suggest that the limited permeability for exogenous MNA, in contrast to that for NA, may be responsible for its lack of suppressor activity against Mvarphi.

Anti-inflammatory effect of 1-methylnicotinamide in contact hypersensitivity to oxazolone in mice; involvement of prostacyclin.

1-methylnicotinamide (MNA) displays anti-inflammatory effects in patients with contact dermatitis, though the mechanisms involved remain unknown. Herein, we examined the anti-inflammatory effects of MNA and its parent molecule, nicotinamide, in the contact hypersensitivity reaction to oxazolone in CBA/J inbred mice. Feeding mice with MNA or nicotinamide (100 mg/kg, 10 days) resulted in the inhibition of the development of contact hypersensitivity reaction by 37% and 35%, respectively, as assessed by the magnitude of ear swelling. This effect was not associated with changes in the expression of adhesion molecules (CD49d(+) and CD54(+)) on CD4(+) and CD8(+) oxazolone-specific T lymphocytes, the major cell component of an inflammatory infiltrate in contact hypersensitivity reaction. Furthermore, in the adoptive transfer model of contact hypersensitivity reaction, pretreatment of mice (recipients of oxazolone-specific T cells), with MNA, resulted in a remarkable anti-inflammatory effect (inhibition of contact hypersensitivity reaction by 66%). Interestingly, in the presence of prostanoid IP receptor antagonist R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO-3244794) (10 mg/kg) the MNA was inactive. In summary, pretreatment with MNA profoundly attenuated contact hypersensitivity reaction in vivo. In particular, the vessel dependent phase of contact hypersensitivity reaction was affected, in spite of the fact that MNA did not alter the expression of adhesive molecules on oxazolone-specific T lymphocytes. However, the anti-inflammatory action of MNA was completely reversed by the antagonist of prostanoid IP receptor. Accordingly, our results demonstrate for the first time that anti-inflammatory properties of MNA are linked to endothelial, PGI(2)-mediated mechanisms.

Anti-inflammatory effects of taurine derivatives (taurine chloramine, taurine bromamine, and taurolidine) are mediated by different mechanisms.

In this study, in an animal model of zymosan-induced peritonitis we have tested anti-inflammatory properties of Taurolidine (TRD), a synthetic derivative of taurine. In vitro, the effect of TRD and HOCl treated TRD on peritoneal macrophages was compared with that of TauCl. We report that locally administered TRD (Taurolin) shows strong anti-inflammatory properties. TRD inhibits vascular permeability increased by inflammatory stimuli; it also significantly attenuates the influx of neutrophils into the peritoneal cavity, as well as the production of pro-inflammatory cytokines (TNF-alpha, IL-6) by peritoneal exudate cells. Chlorination of TRD resulted in the formation of chloramine (TRD-Cl), as confirmed by characteristic UV spectra. Both TRD and TRD-Cl, more effectively than TauCl, inhibited the production of IL-6 by stimulated macrophages. The effect was not dependent on its well-known anti-endotoxin activity since TRD inhibited cytokine production by macrophages stimulated with either LPS or IFN-gamma. Finally, we report that anti-inflammatory activities of TRD and taurine haloamines are mediated by different mechanisms. TRD, in contrast to TauCl and TauBr, does not induce expression of HO-1, a stress inducible enzyme with strong anti-inflammatory properties.

Susceptibility of Propionibacterium acnes and Staphylococcus epidermidis to killing by MPO-halide system products. Implication for taurine bromamine as a new candidate for topical therapy in treating acne vulgaris.

INTRODUCTION: Taurine chloramine (TauCl) and taurine bromamine (TauBr) are the main haloamines produced by activated neutrophils. TauCl exerts both anti-inflammatory and microbicidal activities. Clinical studies showed that TauCl may be useful as an antimicrobial agent in the local treatment of infections. Much less is known about TauBr. Circumstantial evidence suggests that Propionibacterium acnes (PA) has a role in the inflammation of acne. Available topical therapies include antimicrobial agents which reduce total PA numbers and anti-inflammatory agents which suppress activity of the cells present in acne inflammatory lesions. In this study the bactericidal activities of TauBr and TauCl against PA and Staphylococcus epidermidis (SE), as a control strain, were investigated. Moreover, the influence of these haloamines on the generation of reactive oxygen species (ROS) by activated neutrophils was also tested. MATERIALS AND METHODS: TauBr and TauCl were prepared by reaction of taurine with HOBr and HOCl, respectively. The reaction was monitored by UV absorption spectra. The bactericidal activities of TauBr and TauCl were determined by the pourplate method. The generation of ROS by neutrophils was determined by luminol chemiluminescence assay. RESULTS: In our experimental set-up, TauBr showed stronger antibacterial activity than TauCl. Interestingly, PA was significantly more susceptible to TauBr than SE was. Moreover, TauBr at non-cytotoxic concentrations significantly reduced ROS generation by neutrophils. CONCLUSIONS: Since PA is considered to be an etiological agent in acne and ROS are closely correlated with the pathogenesis of inflammatory skin diseases, the reported data suggest that TauBr may be a good candidate for the topical therapy for acne vulgaris.

Influence of macrophage activation on their capacity to bind bacterial antigens studied with atomic force microscopy.

In this work we studied interactions between bacterial antigens and receptors on the surface of macrophages using atomic force microscopy (AFM). We used two bacterial cell wall components: lipopolysaccharide (LPS) derived from gram-negative Escherichia coli and exopolysaccharide (EPS) derived from gram-positive Lactobacillus rhamnosus. Interactions between these bacterial antigens and immune cell receptors were studied in peritoneal macrophages derived from two strains of mice, CBA and C3H/J, in which the Toll-like receptor 4 (TLR4) is genetically disabled. We collected 500 force-distance curves for LPS-activated cells using an EPS-covered AFM tip, and for EPS-activated cells using an LPS-covered AFM tip. Nonactivated cells were tested as reference cells. The results show that LPS-primed macrophages decrease their ability to bind EPS. Surprisingly, EPS-activated macrophages maintain or even increase their ability to bind LPS. This may suggest that in vivo commensal enteric bacteria, such as lactobacilli, will enhance the defense potential of local macrophages against pathogens expressing LPS.

Leukotrienes modulate cytokine release from dendritic cells.

Leukotriene B(4) (LTB(4)) and cysteinyl leukotrienes (CysLTs) are known as potent mediators of inflammation, whereas their role in the regulation of adaptive immunity remains poorly characterized. Dendritic cells (DCs) are specialized antigen-presenting cells, uniquely capable to initiate primary immune responses. We have found that zymosan, but not lipopolysaccharide (LPS) stimulates murine bone marrow-derived dendritic cells (BM-DCs) to produce large amounts of CysLTs and LTB(4) from endogenous substrates. A selective inhibitor of leukotriene synthesis MK886 as well as an antagonist of the high affinity LTB(4) receptor (BLT(1)) U-75302 slightly inhibited zymosan-, but not LPS-stimulated interleukin (IL)-10 release from BM-DCs. In contrast, U-75302 increased zymosan-stimulated release of IL-12 p40 by approximately 23%. Pre-treatment with transforming growth factor-beta1 enhanced both stimulated leukotriene synthesis and the inhibitory effect of U-75302 and MK886 on IL-10 release from DCs. Consistent with the effects of leukotriene antagonists, exogenous LTB(4) enhanced LPS-stimulated IL-10 release by approximately 39% and inhibited IL-12 p40 release by approximately 22%. Both effects were mediated by the BLT(1) receptor. Ligands of the high affinity CysLTs receptor (CysLT(1)), MK-571 and LTD(4) had little or no effect on cytokine release. Agonists of the nuclear LTB(4) receptor peroxisome proliferator-activated receptor-alpha, 8(S)-hydroxyeicosatetraenoic acid and 5,8,11,14-eicosatetraynoic acid, inhibited release of both IL-12 p40 and IL-10. Our results indicate that both autocrine and paracrine leukotrienes may modulate cytokine release from DCs, in a manner that is consistent with previously reported T helper 2-polarizing effects of leukotrienes.

Different pro-inflammatory and immunogenic potentials of Propionibacterium acnes and Staphylococcus epidermidis: implications for chronic inflammatory acne.

INTRODUCTION: Propionibacterium acnes (PA) and Staphyloccocus epidermidis (SE) are two major bacterial strains isolated from acne lesions. Nevertheless, only PA seems to be implicated in the pathogenesis of inflammatory acne vulgaris. Evidence for this, however, remains indirect and the precise role of PA in inflammatory acne is still a matter for conjecture. The aim of this study was to compare some pro-inflammatory and adjuvant properties of PA and SE. MATERIAL/METHODS: To determine some of the pathogenic, immunostimulatory, and pro-inflammatory proper of PA and SE, two experimental models of inflammation were used. In vivo; chronic inflammation was induced by intradermal injection of living bacteria into the ear. In vitro; peritoneal macrophages elicited by the bacteria were examined for their ability to generate reactive oxygen species (ROS), nitric oxide (NO), and cytokines. RESULTS: PA, but not SE, evoked mild local inflammation of infected ears. Macrophages elicited with PA produced more tumor necrosis factor alpha and interleukin IL-12 than those induced with SE, while SE was a stronger inducer of IL-10 production. Both bacteria equally induced the generation of NO and ROS. In contrast, only PA showed adjuvant proper-ties. CONCLUSIONS: The results of these studies indicate that SE, in contrast to PA, does not exert pro-inflammatory properties. Thus it is unlikely that SE may be implicated in the pathogenesis of inflammatory acne vulgaris.

Oxidative modification of type II collagen differentially affects its arthritogenic and tolerogenic capacity in experimental arthritis.

INTRODUCTION: Oxidative modification of proteins affects their biological properties. Previously we have shown that hypochlorite (HOCl), the product of activated neutrophils, enhances protein immunogenecity. Collagen type II, a primary component of cartilage, is commonly used in the induction of arthritis in animals (CIA). The aim of this study was to examine whether HOCl may affect immunogenic, tolerogenic, and arthritogenic properties of collagen. MATERIALS AND METHODS: DBA/J mice were injected with either native (CNAT) or chlorinated collagen (CHOCl) to induce arthritis. The effect of chlorination on collagen properties was measured by evaluation of incidence and severity of CIA. Moreover, the concentration of serum anti-collagen IgG antibodies and myeloperoxidase (MPO) activity in inflamed joints was determined. RESULTS: Mice immunized with CNAT in adjuvant developed arthritis (CIA) with an incidence of 69%. CNAT also exerted tolerogenic properties when injected intravenously either before or shortly after primary immunization, resulting in decreased incidence and severity of CIA, reduced MPO activity in inflamed joints, and lowered serum levels of anti-CNAT IgG anti-bodies. Chlorination of collagen significantly diminished its ability to induce CIA and to trigger generation of anti-CNAT IgG antibodies. Interestingly, chlorination did not affect tolerogenic properties of collagen administered prior to primary immunization with CNAT. CONCLUSIONS: These results suggest that chlorination of collagen may selectively affect functional epitopes of collagen. It is likely that in inflamed joints, neutrophil derived HOCl, in some circumstances, will destroy arthritogenic and immunogenic B cell epitopes, while regulatory T cell epitopes will be preserved.