InVivoMAb anti-mouse CD11b

InVivoMAb anti-mouse CD11b (CLONE: 5C6)

Odobasic D, Kitching AR, Yang Y, O', Sullivan KM, Muljadi RC, Edgtton KL, Tan DS, Summers SA, Morand EF, Holdsworth SR (2013). "Neutrophil myeloperoxidase regulates T-cell-driven tissue inflammation in mice by inhibiting dendritic cell function" Blood 121(20):4195-204. PubMed

Myeloperoxidase (MPO) is important in intracellular microbial killing by neutrophils but extracellularly causes tissue damage. Its role in adaptive immunity and T-cell-mediated diseases is poorly understood. Here, T-cell responses in lymph nodes (LNs) were enhanced by MPO deletion or in vivo inhibition, causing enhanced skin delayed-type hypersensitivity and antigen (Ag)-induced arthritis. Responses of adoptively transferred OT-II T cells were greater in MPO-deficient than wild-type (WT) recipients. MPO, deposited by neutrophils in LNs after Ag injection, interacted with dendritic cells (DCs) in vivo. Culture of murine or human DCs with purified MPO or neutrophil supernatant showed that enzymatically dependent MPO-mediated inhibition of DC activation occurs via MPO-generated reactive intermediates and involves DC Mac-1. Transfer of DCs cultured with WT, but not MPO-deficient, neutrophil supernatant attenuated Ag-specific immunity in vivo. MPO deficiency or in vivo inhibition increased DC activation in LNs after immunization. Studies with DQ-ovalbumin showed that MPO inhibits Ag uptake/processing by DCs. In vivo DC transfer and in vitro studies showed that MPO inhibits DC migration to LNs by reducing their expression of CCR7. Therefore, MPO, via its catalytic activity, inhibits the generation of adaptive immunity by suppressing DC activation, Ag uptake/processing, and migration to LNs to limit pathological tissue inflammation.

Stokes RW, Norris-Jones R, Brooks DE, Beveridge TJ, Doxsee D, Thorson LM (2004). "The glycan-rich outer layer of the cell wall of Mycobacterium tuberculosis acts as an antiphagocytic capsule limiting the association of the bacterium with macrophages" Infect Immun 72(10):5676-86. PubMed

Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen that infects macrophages and other host cells. We show that sonication of M. tuberculosis results in the removal of material from the surface capsule-like layer of the bacteria, resulting in an enhanced propensity of the bacteria to bind to macrophages. This effect is observed with disparate murine and human macrophage populations though, interestingly, not with freshly explanted alveolar macrophages. Enhanced binding to macrophages following sonication is significantly greater within members of the M. tuberculosis family (pathogens) than within the Mycobacterium avium complex (opportunistic pathogens) or for Mycobacterium smegmatis (saprophyte). Sonication does not affect the viability or the surface hydrophobicity of M. tuberculosis but does result in changes in surface charge and in the binding of mannose-specific lectins to the bacterial surface. The increased binding of sonicated M. tuberculosis was not mediated through complement receptor 3. These results provide evidence that the surface capsule on members of the M. tuberculosis family may be an important virulence factor involved in the survival of M. tuberculosis in the mammalian host. They also question the view that M. tuberculosis is readily ingested by any macrophage it encounters and support the contention that M. tuberculosis, like many other microbial pathogens, has an antiphagocytic capsule that limits and controls the interaction of the bacterium with macrophages.

Henderson RB, Hobbs JA, Mathies M, Hogg N (2003). "Rapid recruitment of inflammatory monocytes is independent of neutrophil migration" Blood 102(1):328-35. PubMed

Early neutrophil entry into an inflammatory site is thought to mediate a chemokine switch, inducing subsequent monocyte recruitment through the regulation of monocyte chemoattractant protein-1 (MCP-1) release. As the murine monocyte is poorly characterized and difficult to identify, there has been little examination of either its early recruitment in inflammatory models or of the factors that influence its early migration. The phenotyping of rapidly recruited inflammatory leukocytes with 7/4 and Gr-1 monoclonal antibodies (mAbs) identifies 2 distinct populations, which we characterize as murine monocytes and neutrophils. Monocytes migrate in the first 2 hours of inflammation making use of alpha4beta1 but not of Mac-1 or lymphocyte function-associated antigen-1 (LFA-1) integrins. Early migration is dependent on MCP-1, but neither MCP-1 release nor monocyte recruitment is affected by the reduced neutrophil migration seen in LFA-1-/- mice. Endogenous peritoneal macrophages and mesothelial cells lining the peritoneum contain MCP-1, which is released following thioglycollate stimulation. The murine monocyte therefore responds rapidly to chemokines produced in situ by tissue cells at the site of inflammation with no requirement for prior influx of neutrophils.

Velders GA, Pruijt JF, Verzaal P, van Os R, van Kooyk Y, Figdor CG, de Kruijf EJ, Willemze R, Fibbe WE (2002). "Enhancement of G-CSF-induced stem cell mobilization by antibodies against the beta 2 integrins LFA-1 and Mac-1" Blood 100(1):327-33. PubMed

The beta 2 integrins leukocyte function antigen-1 (LFA-1, CD11a) and macrophage antigen-1 (Mac-1, CD11b) have been reported to play a role in the attachment of CD34(+) cells to stromal cells in the bone marrow. When administered prior to interleukin-8 (IL-8), anti-LFA-1 antibodies completely prevent the IL-8-induced mobilization of hematopoietic stem cells in mice. Here, we studied the role of anti-beta 2 integrin antibodies in granulocyte colony-stimulating factor (G-CSF)-induced mobilization of hematopoietic progenitor cells. Administration of antibodies against the alpha chain of LFA-1 or against the alpha chain of Mac-1 followed by daily injections of G-CSF for more than 1 day resulted in a significant enhancement of mobilization of hematopoietic progenitor cells when compared with mobilization induced by G-CSF alone. Also, the number of late (day 28) cobblestone area-forming cells in vitro was significantly higher after mobilization with anti-LFA-1 antibodies followed by 5 microg G-CSF for 5 days than with G-CSF alone (119 +/- 34 days vs 17 +/- 14 days), indicating mobilization of repopulating stem cells. Pretreatment with blocking antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of LFA-1 and Mac-1, did not result in an effect on G-CSF-induced mobilization, suggesting that the enhancing effect required an interaction of the beta 2 integrins and one of their other ligands. Enhancement of mobilization was not observed in LFA-1-deficient (CD11a) mice, indicating that activated cells expressing LFA-1 mediate the synergistic effect, rather than LFA-1-mediated adhesion.

Dal Canto AJ, Swanson PE, O', Guin AK, Speck SH, Virgin HW (2001). "IFN-gamma action in the media of the great elastic arteries, a novel immunoprivileged site" J Clin Invest 107(2):R15-22. PubMed

Infection of medial smooth muscle cells with gamma-herpesvirus 68 (gammaHV68) causes severe chronic vasculitis that is restricted to the great elastic arteries. We show here that persistence of disease in the great elastic arteries is (a) due to inefficient clearance of viral infection from this site compared with other organs or other vascular sites, and (b) associated with failure of T cells and macrophages to enter the virus-infected elastic media. These findings demonstrate immunoprivilege of the media of the great elastic arteries. We found that IFN-gamma acted on somatic cells during acute infection to prevent the establishment of medial infection and on hematopoietic cells to determine the severity of disease in this site. The immunoprivileged elastic media may provide a site for persistence of pathogens or self antigens leading to chronic vascular disease, a process regulated by IFN-gamma actions on both somatic and hematopoietic cells. These concepts have significant implications for understanding immune responses contributing to or controlling chronic inflammatory diseases of the great vessels.

Lan DT, Makino S, Shirahata T, Yamada M, Nakane A (1999). "Complement receptor type 3 plays an important role in development of protective immunity to primary and secondary Corynebacterium pseudotuberculosis infection in mice" Microbiol Immunol 43(12):1103-6. PubMed

The present study was performed to investigate the role of CR3, the type 3 complement receptor, in host defense against primary and secondary Corynebacterium (C.) pseudotuberculosis infection in mice. Treatment of mice with 5C6, an anti-CR3 monoclonal antibody (mAb), resulted in unrestricted multiplication of bacteria in the organs and dramatically increased mortalities of the infected mice. Histological examinations showed the inflammation, degeneration and necrosis of organs and revealed that the infection-enhancing effect of 5C6 mAb was associated with the failure of mice to focus mononuclear phagocytes at sites of bacterial multiplication. These results suggest that CR3 plays an important role in host defense against primary as well as secondary C. pseudotuberculosis infection in mice.

Avni O, Pur Z, Yefenof E, Baniyash M (1998). "Complement receptor 3 of macrophages is associated with galectin-1-like protein" J Immunol 160(12):6151-8. PubMed

We have previously identified a 16-kDa protein with a pI of 5.1 (P16/5.1) that is associated with macrophage CR3. Microsequencing of P16/5.1 indicated exclusive homology to the beta-galactoside-binding lectin, galectin-1. Abs specific to a galectin-1 unique peptide reacted with P16/5.1. The association of P16/5.1 with CR3 was specifically inhibited by lactose, which binds with high affinity to galectin-1. These data together with similarities in molecular mass and pI suggest that P16/5.1 is galectin-1. Two-color immunofluorescence staining revealed the expression of galectin-1 on the macrophage surface and its colocalization with CR3. However, a surplus of CR3 was free of galectin-1, and some galectin-1 molecules were associated with cell surface receptors other than CR3. Based on these results we propose two models depicting the functional significance of CR3-galectin-1 association: 1) homodimeric galectin-1 possessing a divalent sugar binding site may act as an extracellular adapter molecule that cross-links CR3 with other receptors; and 2) association of galectin-1 with beta-galactosides on the extracellular domain of CR3 may modify the binding affinity of the receptor to its ligand. These possibilities are not mutually exclusive and can clarify the mode by which CR3 transmits signals in macrophages.

Hughes DA, Gordon S (1998). "Expression and function of the type 3 complement receptor in tissues of the developing mouse" J Immunol 160(9):4543-52. PubMed

Macrophage (Mphi) expression of the leukocyte integrins has been implicated in their adhesion and migration in the adult. Little is known, however, of the expression or function of these molecules during development. This study defines the spatial and temporal sequences of expression of the type 3 complement receptor (CR3) in the developing mouse; establishes the functional efficacy of this molecule in spreading, adhesion, and phagocytosis; and investigates its role in inflammatory and constitutive migration. Expression of CR3 on monocytes occurred early compared to Mphi-restricted glycoprotein F4/80, but expression on stellate tissue Mphi appeared later than F4/80 and was transient. Expression of CR3 on resident tissue Mphi is more widespread during development, being retained on only very specific Mphi populations in the adult. Neutrophil polymorphs expressed CR3 from day 17 of gestation onward. The anti-CR3 mAb 5C6 was used to investigate the role of CR3 in adhesion, spreading, and phagocytosis by neonatal Mphi. Neonatal macrophages were found to adhere, spread, and phagocytose by CR3-dependent mechanisms, and a CR3-independent system was implicated in the spreading of neonatal Mphi. The role of CR3 in migration during development was then investigated. 5C6 had potent effects on the early stages of the migration of myelomonocytic cells to an inflammatory stimulus in vivo. Despite efficient transplacental transfer of the Ab from pregnant mother to fetus, the process by which monocytes generate populations of resident tissue Mphi was undisrupted, indicating the existence of CR3-independent mechanisms of monocyte migration during development.

Di Sciullo G, Donahue T, Schachner M, Bogen SA (1998). "L1 antibodies block lymph node fibroblastic reticular matrix remodeling in vivo" J Exp Med 187(12):1953-63. PubMed

L1 is an immunoglobulin superfamily adhesion molecule highly expressed on neurons and involved in cell motility, neurite outgrowth, axon fasciculation, myelination, and synaptic plasticity. L1 is also expressed by nonneural cells, but its function outside of the nervous system has not been studied extensively. We find that administration of an L1 monoclonal antibody in vivo disrupts the normal remodeling of lymph node reticular matrix during an immune response. Ultrastructural examination reveals that reticular fibroblasts in mice treated with L1 monoclonal antibodies fail to spread and envelop collagen fibers with their cellular processes. The induced defect in the remodeling of the fibroblastic reticular system results in the loss of normal nodal architecture, collapsed cortical sinusoids, and macrophage accumulation in malformed sinuses. Surprisingly, such profound architectural abnormalities have no detectable effects on the primary immune response to protein antigens.

Lecoanet-Henchoz S, Plater-Zyberk C, Graber P, Gretener D, Aubry JP, Conrad DH, Bonnefoy JY (1997). "Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18" Eur J Immunol 27(9):2290-4. PubMed

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the alpha chain of the beta 2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activities of murine myeloid cells.

Hammerberg C, Duraiswamy N, Cooper KD (1996). "Reversal of immunosuppression inducible through ultraviolet-exposed skin by in vivo anti-CD11b treatment" J Immunol 157(12):5254-61. PubMed

In both human in vitro models and murine in vivo adoptive transfer studies, UV-induced class II MHC+ CD11b+ leukocytes that infiltrate the epidermis appear to mediate UV-induced immunosuppression. In the present study, their role is further probed using an anti-CD11b mAb (clone 5C6), which is effective in vivo in blocking CD11b+ monocyte/macrophage diapedesis into inflammatory lesions. A single exposure, low dose UV protocol (72 mJ/cm2) that resulted in tolerance only when dinitroflurobenzene was applied 48 h later through the UV-irradiated skin, but not through a distant non-UV-irradiated site, was used. In vivo anti-CD11b treatment in non-UV-irradiated mice did not block contact sensitivity responses. However, the ability to induce a primary contact sensitivity response was completely restored in UV-irradiated mice receiving anti-CD11b. This restoration was associated with partial restoration of papillary dermal class II MHC+ NLDC-145- cells. In vivo anti-CD11b treatment also blocked tolerance induction, which was associated with a 50% reduction in the infiltration of class II MHC+ CD11b+ Gr-1+ monocyte/macrophages into UV-irradiated skin. In addition, anti-CD11b treatment partially protected against epidermal UV injury, in that the epidermal structure was better preserved and the keratinocytes were less severely damaged. CD11b+ leukocytes may thus affect UV-irradiated skin through at least two mechanisms: 1) a class II MHC+ CD11b+ Gr-1+ monocyte/macrophage population inducing a state of tolerance to Ag(s) acquired in UV-irradiated skin, and 2) CD11b+ leukocytes capable of inflicting additional injury to both keratinocytes and constitutive APC damaged by UV photons.

Taylor PC, Chu CQ, Plater-Zyberk C, Maini RN (1996). "Transfer of type II collagen-induced arthritis from DBA/1 to severe combined immunodeficiency mice can be prevented by blockade of Mac-1" Immunology 88(2):315-21. PubMed

Collagen-induced arthritis in susceptible mice is widely accepted as an experimental model for human rheumatoid arthritis (RA). We have investigated the role of the Mac-1 integrin beta 2 in the development and maintenance of arthritis by means of in vivo administration of 5C6 monoclonal antibody (mAb) to block this receptor. Injection of a single dose of 5C6 mAb (0.5 mg, intraperitoneally) prior to the expected onset of collagen-induced arthritis in DBA/1 mice diminished the severity of subsequent disease in these animals, as assessed both clinically and histologically (P < 0.01, chi 2). In the DBA/1 to severe combined immunodeficiency (SCID) transfer model of arthritis, the incidence of clinical arthritis was significantly reduced in SCID mice receiving maintained 5C6 treatment commencing the day prior to administration of donor splenocytes. Histological evaluation of joints from animals without clinically evident arthritis confirmed the absence of an inflammatory infiltrate in 22/27 joints examined. In a minority of these joints, however, synovial hyperplasia was apparent. In contrast, delaying antibody administration until 10 days after donor spleen cell transfer failed to protect three of five SCID recipients. These results confirm a functional role for Mac-1 in the generation of collagen-induced arthritis in mice. Since mAb 5C6 is non-cytotoxic, its action must be by blockade of the interactions between Mac-1 and its natural ligand(s). Our findings support the hypothesis that cells expressing Mac-1 play an important role in the induction and maintenance of joint damage in collagen-induced arthritis.

Whitcup SM, DeBarge LR, Rosen H, Nussenblatt RB, Chan CC (1993). "Monoclonal antibody against CD11b/CD18 inhibits endotoxin-induced uveitis" Invest Ophthalmol Vis Sci 34(3):673-81. PubMed

Purpose: To examine the serial expression of cell adhesion molecules in C3H/HeN mice with endotoxin-induced uveitis, and to study the effect of treatment with a monoclonal antibody against Mac-1 on the development of endotoxin-induced uveitis.

Mielke ME, Rosen H, Brocke S, Peters C, Hahn H (1992). "Protective immunity and granuloma formation are mediated by two distinct tumor necrosis factor alpha- and gamma interferon-dependent T cell-phagocyte interactions in murine listeriosis: dissociation on the basis of phagocyte adhesion mechanisms" Infect Immun 60(5):1875-82. PubMed

Listeria-immune mice are able to express protective immunity in the absence of CD4+ T cells and an apparent granulomatous inflammation. Using a monoclonal antibody (5C6) able to inhibit the recruitment of myelomonocytic cells into inflammatory foci by binding to complement receptor type 3 (CR3/CD11b), we could show that protective immunity and granuloma formation indeed depend on two distinct types of T cell-phagocyte interactions. Listeria-specific CD8+ T lymphocytes, possibly in collaboration with CD4- CD8- T cells, rapidly interact with myelomonocytic cells infiltrating infected tissues in a CR3/CD11b-dependent manner. This interaction results in potent antilisterial protection but not in granuloma formation. On the contrary, CD4+ T cells are able to induce adhesion mechanisms that allow the accumulation of monocytes in granulomatous lesions even in the presence of monoclonal antibody 5C6. However, the protective capacity of these CR3/CD11b-independent T cell-mediated immune mechanisms is low in listeriosis. Tumor necrosis factor alpha and gamma interferon, known to be essential for the expression of both resistance and acquired immunity, are shown to be necessarily involved in granuloma formation, too. It therefore remains to be explained why CD8+ T cells, able to secrete both cytokines, do not induce granuloma formation. The data point to the presence of an as yet undefined CD4+ T cell-derived granuloma-inducing factor and favor the hypothesis that CD8+ T cells, in collaboration with circulating phagocytes, mediate immunity by rapidly liberating listeriae from permissive cells or protecting them from becoming infected.

Rosen H, Gordon S (1987). "Monoclonal antibody to the murine type 3 complement receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo" J Exp Med 166(6):1685-701. PubMed

Macrophage interactions with extracellular matrix and other cells are important in phagocytosis, inflammation, and immunity. To learn more about the surface molecules involved in adhesion we compared the binding of murine macrophages and polymorphonuclear leukocytes (PMN) with artificial substrate in vitro. A distinctive type of adhesion of thioglycollate-elicited peritoneal macrophages (TPM) to bacteriologic plastic (BP) was defined, which was pronase-sensitive, Mg2+-dependent, and required cytoskeletal stabilization. A rat mAb designated 5C6 was isolated because it inhibited TPM attachment to BP, as well as mediating detachment of TPM adherent to that substratum. In addition, it inhibited the attachment of PMN to tissue culture plastic. This antiadhesive property of 5C6 mAb required intact IgG; the F(ab')2 fragment was partially effective and Fab was ineffective. 5C6 recognized the type 3 complement receptor, inhibiting rosetting of EAC3bi to TPM and immunoprecipitating a heterodimer of 160 and 95 kD that comigrated with the M1/70 immunoprecipitate. 5C6 recognized a pronase-stable epitope distinct from that of M1/70. Other mAbs, including M1/70 (CR3) and 2.4G2 (FcR), failed to have any antiadhesive effect in vitro. The inhibitory activity of 5C6 in short-term adhesion assays correlated with its inhibition of recruitment of myelomonocytic cells to a thioglycollate-elicited peritoneal exudate in vivo, after intravenous injection of mAb. 5C6 IgG inhibited recruitment of myelomonocytic cells by 84 +/- 3% at 1 d compared with saline-injected controls. The F(ab')2 fragment and a class-matched control IgG had little effect. Recruitment of TPM at 4 d was also efficiently inhibited by 5C6 IgG. 5C6 IgG was not cytotoxic, had no effect on marrow egress, did not cause increased phagocytic clearance of circulating neutrophils, and had no adverse effect on chemotaxis in vitro. We show that CR3 alone of the LFA-family is necessary for the recruitment of myelomonocytic cells to inflammatory stimuli such as thioglycollate broth. This strategy may be of general use in isolating reagents that inhibit the adhesive function of CR3 and provides a novel approach to antiinflammatory therapy.