ARTHRITIS & RHEUMATISM
Vol. 63, No. 9, September 2011, pp 2651–2660
DOI 10.1002/art.30448
© 2011, American College of Rheumatology
Resolution of Neutrophilic Inflammation by H
2
O
2
in
Antigen-Induced Arthritis
Fernando Lopes, Fernanda M. Coelho, Vivian V. Costa, E
´
rica L. M. Vieira, Lirla ˆndia P. Sousa,
Tarcília A. Silva, Leda Q. Vieira, Mauro M. Teixeira, and Vanessa Pinho
Objective. Neutrophil accumulation contributes
to the pathogenesis of rheumatoid arthritis. This study
was undertaken to examine the ability of H
2
O
2
to
influence neutrophilic inflammation in a model of
antigen-induced arthritis (AIA) in mice.
Methods. AIA was induced by administration of
antigen into the knee joints of previously immunized
mice. Neutrophil accumulation was measured by count-
ing neutrophils in the synovial cavity and assaying
myeloperoxidase activity in the tissue surrounding the
mouse knee joint. Apoptosis was determined by morpho-
logic and molecular techniques. The role of H
2
O
2
was
studied using mice that do not produce reactive oxygen
species (gp91
phox/
mice) and drugs that enhance the
generation or enhance the degradation of H
2
O
2
.
Results. Antigen challenge of immunized mice
induced neutrophil accumulation that peaked at 12–24
hours after challenge. H
2
O
2
production peaked at 24
hours, after which time, the inflammation resolved.
Neutrophil recruitment was similar in wild-type and
gp91
phox/
mice, but there was delayed resolution in
gp91
phox/
mice or after administration of catalase. In
contrast, administration of H
2
O
2
or superoxide dismu-
tase (SOD) resolved neutrophilic inflammation. The
resolution of inflammation induced by SOD or H
2
O
2
was accompanied by an increase in the number of
apoptotic neutrophils. Apoptosis was associated with an
increase in Bax and caspase 3 cleavage and was second-
ary to phosphatidylinositol 3-kinase (PI3K)/Akt
activation.
Conclusion. Our findings indicate that levels of
H
2
O
2
increase during neutrophil influx and are neces-
sary for the natural resolution of neutrophilic inflam-
mation. Mechanistically, enhanced levels of H
2
O
2
(en-
dogenous or exogenous) inhibit p-Akt/NF-B and
induce apoptosis of migrated neutrophils. Modulation
of H
2
O
2
production may represent a novel strategy for
controlling neutrophilic inflammation in the joints.
Rheumatoid arthritis (RA) affects 1% of the
adult population of the world (1). The annual cost of RA
is estimated to be 16 billion dollars, including medical
expenses and indirect costs, such as decreased produc-
tivity and quality of life (2). In RA, the altered balance
between proinflammatory and antiinflammatory cyto-
kine production leads to chronic inflammation, synovio-
cyte proliferation, and erosion of cartilage and bone (3).
Neutrophils are the most abundant leukocyte in the
joints of patients with active RA (4) and are thought to
play a role in the pathogenesis of the disease by inflicting
damage to tissues and releasing proinflammatory cyto-
kines (3,5,6).
More direct evidence of the involvement of neu-
trophils in the pathogenesis of arthritis has come from
studies of animal models of the disease (7). Neutrophils
are attracted into affected joints by chemoattractants
commonly detected in rheumatoid synovial fluid (8). For
example, we have shown that chemokines active on
CXCR1 and CXCR2 receptors play an important role in
driving neutrophil influx and joint damage in experimen-
tal arthritis (6). Accumulation of a particular leukocyte
subset in tissue depends not only on the number of cells
being recruited, but also on the number of cells that are
cleared (by apoptosis) or leave the tissue (9). Recent
Supported by the Conselho Nacional de Desenvolvimento
Cientifico e Tecnolo ´gico, Brazil and the Fundac ¸a ˜o de Amparo a `
Pesquisa do Estado de Minas Gerais, Brazil.
Fernando Lopes, MSc, Fernanda M. Coelho, PhD, Vivian V.
Costa, MSc, E
´
rica L. M. Vieira, MS, Lirla ˆndia P. Sousa, PhD, Tarcı ´lia
A. Silva, PhD, Leda Q. Vieira, PhD, Mauro M. Teixeira, MD, PhD,
Vanessa Pinho, PhD: Universidade Federal de Minas Gerais, Belo
Horizonte, Brazil.
Address correspondence to Vanessa Pinho, PhD, Departa-
mento de Morfologia, Instituto de Cie ˆncias Biolo ´gicas, Universidade
Federal de Minas Gerais, Avenida Anto ˆnio Carlos, 6627 Pampulha,
31270-901 Belo Horizonte, Minas Gerais, Brazil. E-mail:
vpinho@icb.ufmg.br.
Submitted for publication December 21, 2010; accepted in
revised form May 5, 2011.
2651