Karin C. Nabbe

Role of Activatory FcγRI and FcγRIII and Inhibitory FcγRII in Inflammation and Cartilage Destruction during Experimental Antigen-Induced Arthritis

IgG-containing immune complexes, which are found in most RA joints, communicate with hematopoietic cells using three classes of Fc receptors(Fc gamma RI, -II, -III). In a previous study we found that if a chronic T-cell-mediated antigen-induced arthritis (AIA) was elicited in knee joints of FcR gamma-chain-deficient mice that lack functional Fc gamma RI and Fc gamma RIII, joint inflammation was comparable but severe cartilage destruction was absent. We now examined the individual role of the stimulatory Fc gamma RI and Fc gamma RIII and inhibitory Fc gamma RII in inflammation and functional cartilage damage in knee joints with AIA using Fc gamma RI-, Fc gamma RII-, and Fc gamma RIII-deficient mice. Three weeks after immunization with the antigen-methylated bovine serum albumin (BSA), cellular (T-cell responses as measured by lymphocyte proliferation) immunity raised against mBSA was comparable in all groups examined. Humoral (total IgG, IgG1, IgG2a, and IgG2b levels) immunity against mBSA was comparable in Fc gamma RI-/- and Fc gamma RIII-/- but higher in Fc gamma RII-/- if compared to controls. Joint swelling as measured by (99m)Tc uptake at days 1, 3, and 7 was similar in Fc gamma RI-/- and Fc gamma RIII-/- mice and significantly higher in Fc gamma RII-/-. Chronic inflammation and cartilage damage (depletion of proteoglycans, metalloproteinase (MMP)-induced neoepitopes, and matrix erosion) was studied histologically in total knee joint sections stained with hematoxylin or safranin-O. Histologically, at day 7 after AIA induction, exudate and infiltrate in the knee joint was similar in Fc gamma RI-/- and Fc gamma RIII-/- and significantly higher (230% and 340%) in Fc gamma RII-/- mice if compared to controls. Aggrecan breakdown in cartilage caused by MMPs and, which is related to severe irreversible cartilage erosion, was further studied by immunolocalization of MMP-mediated neoepitopes (VDIPEN) and image analysis. MMP-induced neoepitopes determined in various cartilage layers (tibia and femur) were primarily inhibited in Fc gamma RI-/- (79 to 87% and 87 to 88%, respectively) and comparable in Fc gamma RIII-/-. VDIPEN neoepitopes were much higher (82 to 122% and 200 to 250%, respectively) in Fc gamma RII-/- mice. Initial depletion of proteoglycans was similar (60 to 100%) in all groups. In the chronic phase, cartilage matrix erosion in the lateral and medial tibia was significantly elevated in Fc gamma RII-/- (222% and 186%, respectively) but not in Fc gamma RI-/- or Fc gamma RIII-/- mice. These results suggest that during T-cell-mediated AIA, Fc gamma RI and Fc gamma RIII act in concert in acute and chronic inflammation whereas Fc gamma RI is the dominant FcR involved in severe cartilage destruction. Fc gamma RII is a crucial inhibiting factor in acute and chronic inflammation and cartilage erosion.

The Inhibitory Receptor FcγRII Reduces Joint Inflammation and Destruction in Experimental Immune Complex-Mediated Arthritides Not Only by Inhibition of FcγRI/III but Also by Efficient Clearance and Endocytosis of Immune Complexes

Studies of FcgammaRII-/- mice identified the inhibitory function of this receptor in joint inflammation and cartilage destruction induced with immune complexes (ICs). To extend our insight in the role of FcgammaRII in arthritis, we explored the role of FcgammaRII in the absence of activating receptors I and III using FcgammaRI/III-/- as well as FcgammaRI/II/III-/- mice. When antigen-induced arthritis (AIA) was elicited, which is a mixture of T cell and IC-driven inflammation, arthritis was almost absent at day 7 in FcgammaRI/III-/- mice. Remarkably, in FcgammaRI/II/III-/- mice, this model induced a tremendously increased arthritis as compared to wild-type controls. This implies that FcgammaRII regulates joint inflammation also in the absence of activating FcgammaRI and III. To confirm the IC specificity of this finding, similar studies were done with ICs or zymosan as arthritogenic stimuli. Strongly elevated inflammation was found in FcgammaRI/II/III-/- mice with IC but not with zymosan. Clearance studies identified accumulation of IgG in the knee joint in the absence of FcgammaRII. Moreover, macrophages expressing only FcgammaRII showed prominent endocytosis of preformed soluble ICs not different from controls. In total absence of FcgammaR (FcgammaRI/II/III-/-), macrophages completely failed to endocytose ICs. Although joint inflammation was much higher in AIA arthritic knee joints of FcgammaRI/II/III-/- and the inflammatory cells still expressed an inflammatory phenotype, severe cartilage destruction (MMP-mediated neoepitopes in the matrix and chondrocyte death) was completely prevented in contrast to the marked destruction which was observed in the wild-type. Our study indicates that FcgammaRII reduces joint inflammation in the absence of activating FcgammaR by promoting endocytosis and clearance of ICs from the joint. Infiltrating cells, which fail to express activating FcgammaR although they still become stimulated are no longer capable of inducing severe cartilage destruction.

Local IL-13 gene transfer prior to immune-complex arthritis inhibits chondrocyte death and matrix-metalloproteinase-mediated cartilage matrix degradation despite enhanced joint inflammation

During immune-complex-mediated arthritis (ICA), severe cartilage destruction is mediated by Fcγ receptors (FcγRs) (mainly FcγRI), cytokines (e.g. IL-1), and enzymes (matrix metalloproteinases (MMPs)). IL-13, a T helper 2 (Th2) cytokine abundantly found in synovial fluid of patients with rheumatoid arthritis, has been shown to reduce joint inflammation and bone destruction during experimental arthritis. However, the effect on severe cartilage destruction has not been studied in detail. We have now investigated the role of IL-13 in chondrocyte death and MMP-mediated cartilage damage during ICA. IL-13 was locally overexpressed in knee joints after injection of an adenovirus encoding IL-13 (AxCAhIL-13), 1 day before the onset of arthritis; injection of AxCANI (an empty adenoviral construct) was used as a control. IL-13 significantly increased the amount of inflammatory cells in the synovial lining and the joint cavity, by 30% to 60% at day 3 after the onset of ICA. Despite the enhanced inflammatory response, chondrocyte death was diminished by two-thirds at days 3 and 7. The mRNA level of FcγRI, a receptor shown to be crucial in the induction of chondrocyte death, was significantly down-regulated in synovium. Furthermore, MMP-mediated cartilage damage, measured as neoepitope (VDIPEN) expression using immunolocalization, was halved. In contrast, mRNA levels of MMP-3, -9, -12, and -13 were significantly higher and IL-1 protein, which induces production of latent MMPs, was increased fivefold by IL-13. This study demonstrates that IL-13 overexpression during ICA diminished both chondrocyte death and MMP-mediated VDIPEN expression, even though joint inflammation was enhanced.

NADPH-oxidase-driven oxygen radical production determines chondrocyte death and partly regulates metalloproteinase-mediated cartilage matrix degradation during interferon-γ-stimulated immune complex arthritis

In previous studies we have found that FcγRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-γ-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcγRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox-/- mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox-/- mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-γ was overexpressed by injection of adenoviral IFN-γ in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-γ-stimulated arthritic p47phox-/- mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox-/- mice at both time points. FcγRI mRNA expression was significantly lower, and FcγRII and FcγRIII were higher, in p47phox-/- mice than in controls. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and aggravates MMP-mediated cartilage destruction during IFN-γ-stimulated IC-mediated arthritis. Upregulation of FcγRI by oxygen radicals may contribute to cartilage destruction.

FcγRI Up-Regulation Induced by Local Adenoviral-Mediated Interferon-γ Production Aggravates Chondrocyte Death during Immune Complex-Mediated Arthritis

Using various FcγR-deficient mice, we have obtained suggestive evidence that FcγRI on macrophages is responsible for severe cartilage destruction during arthritis mediated by immune complexes (ICs). This role of FcγRI is pronounced in the presence of activated Th1 cells and a likely Th1 cell-derived cytokine mediating up-regulation of FcγRI expression is interferon (IFN)-γ. We now investigated whether local overexpression of IFN-γ using an adenoviral vector is able to elevate cartilage destruction during experimental immune complex-mediated arthritis (ICA) and to what extent this process is FcγRI-mediated. IFN-γ overexpression during ICA had no significant effect on the total cell mass infiltrating the knee joint. However, a higher percentage of macrophages expressing markers for a proinflammatory phenotype was found and these macrophages were situated in close proximity of the cartilage surface. Interestingly, cartilage destruction as studied by matrix metalloproteinase (MMP)-mediated proteoglycan damage (VDIPEN expression), chondrocyte death, and erosion was significantly increased. This effect of IFN-γ was only found in the presence of ICs, as IFN-γ overexpression during zymosan-induced arthritis, which is not IC-dependent, did not lead to severe cartilage destruction. These results imply a crucial role for ICs and the IgG-binding receptors in the aggravation of cartilage damage by IFN-γ. Local overexpression of IFN-γ induced increased FcγRI mRNA levels in synovium. To study whether this up-regulation of FcγRI mediates aggravation of cartilage destruction, ICA was raised in FcγRI −/− and their wild-type controls. IFN-γ resulted in elevated VDIPEN expression, which was still present in FcγRI −/− . Of great interest, chondrocyte death remained low in FcγRI −/− . These results indicate that IFN-γ overexpression deteriorates cartilage destruction in the presence of ICs and that FcγRI is crucial in the development of chondrocyte death.

Safety of glucocorticoids can be improved by lower yet still effective dosages of liposomal steroid formulations in murine antigen-induced arthritis: comparison of prednisolone with budesonide

UNLABELLED The goal of this study was to compare the effects of liposomal and free glucocorticoid formulations on joint inflammation and activity of the hypothalamic-pituitary-adrenal (HPA) axis during experimental antigen-induced arthritis (AIA). A dose of 10mg/kg liposomal prednisolone phosphate (PLP) gave a suppression of the HPA-axis, as measured by plasma corticosterone levels in mice with AIA and in naïve mice. In a subsequent dose-response study, we found that a single dose of 1mg/kg liposomal prednisolone phosphate (PLP) was still equally effective in suppressing joint inflammation as 4 repeated once-daily injections of 10mg/kg free PLP. Moreover, the 1mg/kg liposomal PLP dose gave 22% less suppression of corticosterone levels than 10mg/kg of liposomal PLP at day 14 of the AIA. In order to further optimize liposomal glucocorticoids, we compared liposomal PLP with liposomal budesonide phosphate (BUP) (1mg/kg). At 1 day after treatment, liposomal BUP gave a significantly stronger suppression of joint swelling than liposomal PLP (lip. BUP 98% vs. lip. PLP 79%). Both formulations also gave a strong and lasting suppression of synovial infiltration in equal amounts. However, at day 21 after AIA, liposomal PLP still significantly suppressed corticosterone levels, whereas this suppression was not longer statistically significant for liposomal BUP. CONCLUSION Liposomal delivery improves the safety of glucocorticoids by allowing for lower effective dosing. The safety of liposomal glucocorticoid may be further improved by encapsulating BUP rather than PLP.

Dendritic cells from patients with rheumatoid arthritis lack the interleukin 13 mediated increase of Fc gamma RII expression, which has clear functional consequences.

Background: Dendritic cell (DC) function is largely tailored by Fc gamma receptors (FcγR) and is critical for every immune response. Objective: To compare interleukin (IL) 13 mediated regulation of FcγRII and its related DC function between healthy controls and patients with rheumatoid arthritis (RA). Methods: DC were derived from peripheral blood mononuclear cells according to standardised protocols. FcγRI, II, and III expression and DC phenotype were assessed by FACS analysis. The level of cytokine production and chemokine expression was measured by Luminex and real time quantitative polymerase chain reaction techniques. Antigen uptake capacity was studied by DC fluorescent heat aggregated immunoglobulins and FACS analysis. Results: Replacement of IL4 by IL13 clearly increased the expression of FcγRII on DC from healthy controls (CDC), but had no effect on DC from patients with RA (RADC). The lower production of inflammatory mediators by IL13 CDC upon FcγR mediated triggering suggests that IL13 induces up regulation of specifically FcγRII. RADC co-cultured with IL4 already displayed an inhibitory DC phenotype, but this inhibitory phenotype was not augmented by the addition of IL13. The defective FcγRII regulation was further substantiated by the finding that IL13 CDC increased antigen uptake capacity, whereas IL13 RADC did not. Conclusion: IL13 regulates the expression of inhibitory FcγRII in normal subjects but not in RA, potentially resulting in a chronic proinflammatory immune reaction in RA. Unravelling the underlying mechanisms of FcγRII regulation might lead to new therapeutic targets in RA.

FcγRI up-regulation induced by local adenoviral-mediated IFN-γ production aggravates chondrocyte death during immune-complex-mediated arthritis

Using various FcγR-deficient mice, we have obtained suggestive evidence that FcγRI on macrophages is responsible for severe cartilage destruction during arthritis mediated by immune complexes (ICs) and Th1 cells. In contrast, in arthritis mediated solely by ICs, FcγRIII seems more important. This suggests that T-cell-mediated FcγRI up-regulation promotes pronounced cartilage destruction. A likely Th1-cell-derived cytokine mediating FcγRI expression is interferon-γ (IFN-γ). In the present study we investigated whether IFN-γ is able to up-regulate cartilage destruction during experimental immune complex-mediated arthritis (ICA) and, if so, whether this mechanism is indeed regulated by FcγRI. IFN-γ was locally overexpressed in the murine knee joint prior to ICA induction by the use of adenoviral vectors. This had no significant effect on joint inflammation as studied by histology. However, irreversible cartilage destruction as studied by the degree of chondrocyte death was markedly enhanced. IFN-γ overexpression resulted in a fivefold increase in chondrocyte death, in comparison with the control group, which had received a control adenoviral vector expressing GFP (AdGFP). To study whether this effect of IFN-γ was related to the presence of ICs, IFN-γ was also overexpressed in a naive joint and during zymosan-induced arthritis, which is an IC-independent arthritis model. No severe cartilage destruction was found, implying a crucial role for ICs and their receptors (FcγRs) in the IFN-γ effect. When IFN-γ was overexpressed in murine knee joints, FcγRI mRNA expression was up-regulated in synovial cells. To prove that the aggravation of chondrocyte death by IFN-γ is indeed FcγRI-mediated, ICA was raised in FcγRI-/-. IFN-γ overexpression did not result in significant elevation of joint inflammation either in FcγRI-/- or their wild-type controls. Interestingly, although IFN-γ was overexpressed, chondrocyte death remained absent in FcγRI-/-, whereas in wild-type controls chondrocyte death was highly increased after IFN-γ overexpression. These results indicate that IFN-γ can aggravate cartilage destruction in an IC-dependent fashion, mediated by FcγRI.

Activating FcγRIII determines cartilage destruction during immune complex arthritis but not in the presence of T-cell immunity

We have recently shown that activating Fcγ receptors determine metalloproteinase (MMP)-induced cartilage destruction, seen in various murine models of arthritis mediated by immune complexes (IC). In the mouse, two activating FcγR (FcγRI and FcγRIII) which bind IC have been described. In this study, we investigated the role of activating FcγRIII in MMP-mediated cartilage destruction in two different models of experimental arthritis, one induced only by ICs and the second by ICs and T cells.