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. 2010 Mar;62(3):753-64.
doi: 10.1002/art.27238.

Neutrophils in a mouse model of autoantibody-mediated arthritis: critical producers of Fc receptor gamma, the receptor for C5a, and lymphocyte function-associated antigen 1

Paul A Monach  1 , Peter A Nigrovic, Mei Chen, Hanno Hock, David M Lee, Christophe Benoist, Diane Mathis
Affiliations

Neutrophils in a mouse model of autoantibody-mediated arthritis: critical producers of Fc receptor gamma, the receptor for C5a, and lymphocyte function-associated antigen 1

Paul A Monach et al. Arthritis Rheum. 2010 Mar.

Abstract

Objective: Neutrophils represent a prominent component of inflammatory joint effusions and are required for synovial inflammation in mouse models, but the mechanisms are poorly understood. In this study, we developed a system with which to test the importance of the production of specific factors by neutrophils in a mouse model of arthritis.

Methods: Neutrophil-deficient Gfi-1(-/-) mice were administered sublethal doses of radiation and were then engrafted with donor bone marrow cells (BMCs), which resulted in the production of mature neutrophils within 2 weeks. By reconstituting with BMCs from mice lacking selected proinflammatory factors, we generated mice that specifically lacked these factors on their neutrophils. Arthritis was initiated by transfer of K/BxN serum to identify the role of defined neutrophil factors on the incidence and severity of arthritis.

Results: Neutrophils lacking the signaling chain of stimulatory Fc receptors (FcRgamma(-/-)) were unable to elicit arthritis, but neutrophils lacking FcgammaRIII still did so. Neutrophils lacking the chemotactic or adhesion receptor C5a receptor (C5aR) or CD11a/lymphocyte function-associated antigen 1 (LFA-1) also failed to initiate arthritis but could enter joints in which inflammation had been initiated by wild-type neutrophils. Neutrophils unable to produce interleukin-1alpha (IL-1alpha) and IL-1beta (IL-1alpha/beta(-/-)) or leukotrienes (5-lipoxygenase [5-LOX(-/-)]) produced arthritis of intermediate severity. The inability of neutrophils to make tumor necrosis factor or to express receptors for tumor necrosis factor or IL-1 had no effect on arthritis.

Conclusion: A novel transfer system was developed to identify neutrophil production of FcRgamma, C5aR, and CD11a/LFA-1 as critical components of autoantibody-mediated arthritis. Neutrophil production of IL-1 and leukotriene B(4) likely contributes to inflammation but is not essential. Molecular requirements for neutrophil influx into joints become more permissive after inflammation is initiated.

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Figures

Figure 1
Figure 1
A. Resistance of Gfi-1−/− mice to arthritis induction by K/BxN serum. The maximum severity in each of 25 Gfi-1−/− mice is shown; bar = mean +/− SEM. B. Restoration of arthritis susceptibility in Gfi-1−/− mice by daily (days 0–3) injection of BMC. Results from four reconstituted mice and three Gfi-1−/− controls are shown. C–E. Restoration of circulating neutrophils, and susceptibility to arthritis, by BMC transfer into Gfi-1−/− mice. C. Gfi-1−/− mice were sub-lethally irradiated, then reconstituted with BMC from wild-type CD45.1 congenic donors. Circulating neutrophils were quantified over time. Results from four mice are shown, as is mean +/− SD of three wild-type mice (WT45.1) for comparison. D. Ankle tissue from reconstituted mice was stained for CD11b (a marker of macrophage-like synoviocytes) and either CD45.1 or CD45.2. CD11b co-localized with host-derived CD45.2 (right) but not donor-derived CD45.1 (left). E. Arthritis was induced in reconstituted Gfi-1−/− mice (Gfi-1 + WT) and monitored by clinical index and change in ankle thickness. Gfi-1−/− (as in panel A) and Gfi-1+/+ (WT) littermates served as controls. Points indicate means ± SEM, P < 0.001 between each pair of groups.
Figure 2
Figure 2
Arthritis severity in mice with neutrophils lacking IL-1, TNF, or their receptors. Gfi-1 −/− mice were sub-lethally irradiated, then reconstituted with lineage-depleted bone-marrow cells from donor mice lacking expression of IL-1α and β , TNF, IL-1RI or TNFRI and II, or from control (WT) mice. Arthritis was induced by K/BxN serum transfer starting 2 weeks after reconstitution, and incidence (clinical index ≥3 on multiple occasions) and severity (clinical index 0–12 or change in ankle thickness) were followed over the next 1.5 – 2 weeks. Unmanipulated Gfi-1−/−mice (Gfi–1) served as controls in some experiments. Each point indicates mean severity among all (4–8) arthritic mice in a group, with error bars indicating SEM. Comparing groups at 1.5 weeks after serum transfer: P < 0.01 comparing Gfi+IL-1αβ with Gfi-1+WT, P > 0.05 for other comparisons.
Figure 3
Figure 3
Arthritis induction in mice with neutrophils lacking 5-LO. Gfi-1−/− mice were sub-lethally irradiated, then reconstituted with bone-marrow cells from donor mice lacking expression of 5-LO, or from control (WT) mice. Arthritis was induced by serum transfer 2 weeks after reconstitution, and incidence (clinical index ≥3 on multiple occasions) and severity (clinical index 0–12 or change in ankle thickness) were followed for the next 2 weeks. Each point indicates mean severity in all (6–7) mice in a group, with error bars indicating SEM. Comparing groups at 1.5 weeks after serum transfer: P = 0.047 comparing Gfi-1+5-LO with Gfi-1+WT.
Figure 4
Figure 4
Arthritis induction in mice with neutrophils lacking stimulatory Fc receptors. Gfi-1−/− mice were sub-lethally irradiated, then reconstituted with bone-marrow cells from donor mice lacking expression of either FcRγ (FcRg) or Fcγ RIII (FcgRIII), or from control (WT) mice. Unmanipulated Gfi-1−/− mice (Gfi-1) served as controls in some experiments. Arthritis was induced by serum transfer 2 weeks after reconstitution, and incidence (clinical index ≥3 on multiple occasions) and severity (clinical index 0–12 or change in ankle thickness) were followed for the next 2 weeks. Each point indicates mean severity among all (4–6) mice in a group, with error bars indicating SEM. Comparing groups at 1.5 weeks after serum transfer: P < 0.001 comparing Gfi-1+FcRγ with Gfi-1+WT, P > 0.05 comparing Gfi-1+Fcγ RIII with Gfi-1+WT.
Figure 5
Figure 5
Arthritis induction in mice with neutrophils lacking C5aR or CD11a/LFA-1, and influx of these mutant neutrophils into inflamed joints. A. Gfi-1−/− mice were sub-lethally irradiated, then reconstituted with bone-marrow cells from donor mice lacking expression of either C5aR or CD11a (LFA-1), or from control (WT) mice. Arthritis was induced by serum transfer 2 weeks after reconstitution, and incidence (clinical index ≥3 on multiple occasions) and severity (clinical index 0–12 or change in ankle thickness) were followed for the next 2 weeks. Each point indicates mean severity among all (4–7) mice in a group, with error bars indicating SEM. Comparing groups at 1.5 weeks after serum transfer: P < 0.001 comparing either Gfi-1+C5aR or Gfi-1+CD11a/LFA-1 with Gfi-1+WT. B. Gfi-1−/− mice were sub-lethally irradiated, then reconstituted with mixtures of wild-type (CD45.1) and mutant (CD45.2) bone-marrow cells. Blood and synovial fluid were harvested from arthritic mice 2–13 days after serum transfer, and the percentages of neutrophils of wild-type and mutant origin were determined by flow cytometry. Groups of odds ratios obtained in multiple trials were compared by ANOVA and Tukey-Kramer multiple comparisons test: p < 0.01 comparing CD11a−/− with either C5aR−/− or FcRγ −/−, p = NS comparing C5aR−/− with FcRγ −/−
Figure 6
Figure 6
Model of arthritis induction by K/BxN serum, with emphasis on role of neutrophils. The phase of early vascular leak (shown by extravasation of a fluorescent probe 10 minutes after serum injection) requires neutrophils and mast cells, but neutrophil requirements are unclear, since stimulatory FcR's must only be expressed on radio-resistant cells. For inflammation to be initiated at clinically-detectable levels, neutrophils require the expression of stimulatory FcR's, C5aR, CD11a/LFA-1, and BLT-1, and mast cells require expression of stimulatory FcR's, C5aR, and IL-1, based on the current and previously-published work. Once inflammation is established in otherwise-resistant mice (e.g., by wild-type neutrophils or injection of exogenous IL-1), then neutrophils may still enter the joint space despite lacking expression of the individual receptors required to initiate disease. Macrophages derived from circulating monocytes are likely a major source of IL-1 and TNF at this point, but this is uncertain. Photos of vascular leak appear courtesy of B. Binstadt; drawings of leukocytes were copied from www.wikipedia.org.
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