Depletion of synovial macrophages in rheumatoid arthritis by an anti-FcgammaRI-calicheamicin immunoconjugate

Abstract

Background: Monocytes/macrophages have an important and versatile role in joint inflammation and destruction in rheumatoid arthritis (RA).

Objective: To determine the efficiency of monocyte/macrophage elimination by a new drug conjugated antibody (CD64-calicheamicin (CD64-CaMi)) directed to the high affinity receptor for IgG (FcgammaRI).

Methods: Mononuclear cells from peripheral blood and synovial fluid of patients with RA were cultured in the presence of CD64-CaMi. Cell death of monocytes/macrophages was measured by analysis of phenotypic changes (light scatter patterns, CD14 expression, and FcgammaRI expression) and nuclear DNA fragmentation. The selectivity of CD64-CaMi was checked by using FcgammaRI deficient and FcgammaRI transfected cell lines. In addition, the indirect effect of CD64-CaMi-induced macrophage cell death on arthritogenic T(h1) cell activity was determined.

Results: Inflammatory macrophages from RA synovial fluid, expressing increased FcgammaRI levels, were efficiently killed by CD64-CaMi through induction of DNA fragmentation. CD64-CaMi-induced cell death of monocytes/macrophages from peripheral blood of patients with RA proved less efficient. Induction of synovial macrophage death by CD64-CaMi was accompanied by efficient inhibition of proinflammatory T(h1) cytokine production.

Conclusion: Together, the presented data suggest that elimination of macrophages through a new FcgammaRI directed CD64-CaMi is feasible. Because monocytes from peripheral blood are also eliminated by this immunoconjugate, additional experimental studies should validate its potential for local (intra-articular) application in the treatment of RA.

Figure 1

CD64-CaMi induces FcγRI dependent cell death of FcγRI transfected, but not FcαRI transfected, IIA1.6 cells. Proliferation of IIA1.6 cells (2x10⁵/ml) was measured by [³H]thymidine incorporation (proliferation) after 3 days of culture (n = 3). The effects of CD64-CaMi were expressed as percentages of control cultures in the absence of immunoconjugate. *Significant difference of p<0.05.

Figure 2

Flow cytometric analysis of the effect of CD64-CaMi on PB monocytes and SF macrophages from patients with RA. (A) Representative FcγRI/CD14 staining of MC from PB and SF cultured for 24 hours with or without CD64-CaMi. CD64-CaMi-induced death of monocytic cells was associated with a reduction in CD14⁺ monocytic cells from 22.9% to 14.0% (almost 40% reduction). (B) Average of CD64-CaMi-induced monocyte/macrophage cell death from PB and SF (both n = 6) are expressed as a decrease in viable cells compared with controls, measured as a reduction in CD14 expression after 24 hours of culture. *Significant differences of CD64-CaMi-induced cell death compared with control cultures (p<0.05). At a CD64-CaMi concentration of 10^–9 mol/l, cell death of macrophages from SF, measured by reduction in CD14+ cells (mean (SD) 57 (7)%), was significantly higher than in PB (19 (3)%; †p<0.05).

Figure 3

Induction of nuclear DNA fragmentation of CD68⁺ cells by CD64-CaMi. DNA content of CD68^– lymphocytic and CD68⁺ monocytic cells was stained with PI and measured by flow cytometry (FACS). (A) Representative analysis showing that incubation of SFMC with CD64-CaMi (10^–8 mol/l for 24 hours) results in a strong increase in apoptotic CD68⁺ cells with reduced nuclear DNA content owing to DNA fragmentation (from 3.5% to 92.1% of CD68⁺ cells). (B) On average (n = 3, 10^–8 mol/l for 24 hours), a mean (SD) change from 4.2 (0.2)% apoptotic macrophages in control culture to 57.5 (18.5)% upon culture with CD64-CaMi (p<0.05) was seen. Apoptotic cell death of CD68^– lymphocytic cells was not significantly changed (8.2 (4.0)% to 13.1 (8.9)%). *Significant cell death of CD68⁺ cells (p<0.05).

Figure 4

CD64-CaMi-induced macrophage apoptosis in time prevents CD3/CD28-induced T cell cytokine secretion (n = 3). SFMC were cultured with CD64-CaMi at a concentration of 10^–8 mol/l and 10^–9 mol/l or without CD64-CaMi for 3 days. After this, T cells were costimulated by CD3/CD28 for 24 hours, and IFNγ and TNFα levels were measured. *Significant inhibitions of T cell activity upon (pre-) treatment with CD64-CaMi compared with control cultures (p<0.05).