Effects of CTLA4-Ig on human monocytes
Tono et al. Inflammation and Regeneration
Effects of CTLA4-Ig on human monocytes
Toshihiro Tono 0
Satoko Aihara 0
Takayuki Hoshiyama 0
Yoshiyuki Arinuma 0
Tatsuo Nagai 0
Shunsei Hirohata 0
0 Department of Rheumatology and Infectious Diseases, Kitasato University School of Medicine , 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374 , Japan
Background: Abatacept, a CTLA4-Ig fusion protein attenuates T cell activation by inhibiting the CD80/86-CD28 costimulatory pathway that is required for the proper T cell activation and thus displays beneficial effects in the treatment of rheumatoid arthritis (RA). Although some studies have disclosed the in vitro effects of this biological agent on the immune-competent cells, the precise mechanisms of action in RA still remain unclear. The current studies were therefore undertaken to explore the effects of abatacept on monocytes in detail. Methods: Monocytes from healthy donors were cultured in the presence of staphylococcal enterotoxin B (SEB) with pharmacologically attainable concentrations of abatacept or control IgG-Fc. The expression of CD80 and CD86 and the induction of apoptosis of monocytes were measured by flow cytometry. The expression of CD80 and CD86 messenger RNA (mRNA) was determined by quantitative RT-PCR. Results: Abatacept promoted apoptosis of SEB-stimulated monocytes. The induction of apoptosis of monocytes by these biological agents was reversed by the addition of IgG, but not IgG-F(ab′)2 fragments. Furthermore, abatacept significantly suppressed the expression of CD80, but not that of CD86 at protein levels. Finally, abatacept significantly suppressed the expression of mRNA for CD80 of monocytes stimulated with SEB, but not that of CD86. Conclusions: These results demonstrate that one of the mechanisms of action of abatacept involves the induction of apoptosis of monocytes, which involves interaction with Fc receptor on monocytes. Moreover, the data also demonstrate that abatacept selectively suppresses the expression of CD80 at mRNA levels.
Abatacept; Monocytes; Apoptosis; Costimulation molecules
Background
Abatacept, a CTLA4-Ig fusion protein, attenuates T cell
activation by inhibiting the CD80/CD86–CD28
costimulatory pathway that is required for the proper T cell activation
and thus displays beneficial effects in the treatment of
rheumatoid arthritis (RA) [
1
]. Although some studies have
disclosed the in vitro effects of this biological agent on the
immune-competent cells [
1
], the precise mechanisms of
action in RA still remain unclear.
CTLA4-Ig has been suggested to display some effects
other than inhibition of T cells. Thus, reverse signaling
to dendritic cells upon binding of CTLA4-Ig to CD80/
CD86 has been shown to interfere with dendritic cell
activation and function [
2, 3
]. Since monocytes express
CD80/CD86 as well [4], it is possible that abatacept
might influence the function of monocytes. The current
studies were therefore undertaken to explore the effects
of abatacept on monocytes in detail. Special attention
was paid to the capacities of this biological agent to
induce apoptosis of monocytes and to modulate the
expression of costimulation molecules.
Methods
Monoclonal antibodies
A variety of monoclonal antibodies (mAbs) were used in
this study, including fluorescein isothiocyanate
(FITC)conjugated anti-CD80 (Immunotech, Marseille, France),
FITC-conjugated anti-CD86 (Ancell, Bayport, MN), and
FITC-conjugated control mouse IgG1 (Dako, Glostrup,
Denmark).
Cell preparation
Peripheral blood mononuclear cells (PBMCs) were
obtained from healthy adult volunteers who gave informed
consent, by centrifugation of heparinized venous blood
over sodium diatrizorate-Ficoll gradients. Monocytes
were prepared from PBMC using Monocyte Isolation Kit
II (Miltenyi Biotec). Monocyte population obtained in
this manner contained < 0.1% CD3+ cells, < 0.1% CD19+
cells, and > 93% CD14+ cells.
Reagents
Abatacept was purchased from Bristol-Myers Squibb
(Tokyo). Control human IgG1 was purified from serum
of a patient with human IgG1 myeloma using
DEAESepharose column. Human IgG-Fc and human IgG-F(ab′)2
(Gamma-Venin® P) were purchased from MP Biomedicals,
Santa Ana, CA, and Sanofi, Paris, France, respectively.
Cell cultures
RPMI 1640 medium (Nikken, Kyoto, Japan) supplemented
with penicillin G (100 U/ml) (Life Technologies, Grand
Island, NY), streptomycin (100 μg/ml) (Life Technologies),
L-glutamine (0.3 mg/ml) (Sigma-Aldrich, St Louis, MO),
and 10% fetal bovine serum (JRH Bio Sciences, Lenexa, KS)
were used for cultures. Purified monocytes (1 × 106/well)
were cultured in the presence of staphylococcal enterotoxin
B (SEB) (100 pg/ml) (Serva, Heidelberg, Germany) in each
well of 24-well flat-bottomed microtiter plates (Nunc,
Roskilde, Denmark) with control IgG (100 μg/ml), IgG-Fc
(100 μg/ml), or abatacept (100 μg/ml) for 24 or 48 h. SEB
stimulation and the addition of abatacept or IgG were
simultaneous. Also, purified monocytes (1 × 106 (...truncated)