REVIEW ARTICLE

Role of peroxynitrite-modified biomoleculesin the etiopathogenesis of systemic lupus erythematosus

Rizwan Ahmad • Haseeb Ahsan

Received: 24 July 2012 / Accepted: 6 November 2012 / Published online: 23 November 2012

� Springer-Verlag Italia 2012

Abstract Systemic lupus erythematosus (SLE) is a

chronic inflammatory autoimmune disease characterized

by autoantibodies directed against various biomolecules.

The initial immunogens that drive the development of SLE

are unknown, but characteristics of the immune response in

SLE suggest that it is an antigen-driven response, and a

chromatin antigen could be one of the immunogens for the

production of antinuclear antibodies (ANA) in SLE. Other

factors implicated in the pathogenesis of SLE include

nitrogen-free radicals such as nitric oxide and peroxyni-

trite. The free radical-mediated damage to proteins results

in the modification of amino acid residues, cross-linking of

side chains and fragmentation. The tyrosine residues in

proteins are susceptible to attack by various reactive

nitrogen intermediates, including peroxynitrite to form

3-nitrotyrosine (3-NT). The presence of nitrated proteins

in vivo indicates that peptides derived from the proteolytic

degradation of modified proteins could serve as neoanti-

gens. Histones are highly conserved proteins that are rich in

basic amino acids lysine and arginine. Autoantibodies

against histones and anti-DNA antibodies are present in

SLE. The anti-DNA autoantibodies coexist with anti-his-

tone autoantibodies and may react with chromatin-associ-

ated histones and histone complexes. Elevated levels of

reactive nitrogen species (RNS) in SLE patients suggest a

possible role in the pathogenesis of the disease. The

alteration of proteins resulting from photomodification or

peroxynitrite could lead to the development of antibodies.

Therefore, the modified proteins or photoadducts could

have important implications in autoimmunity, and under-

standing the pathophysiology of peroxynitrite-modified

biomolecules could lead to a better understanding of

autoimmune phenomenon in SLE.

Keywords Peroxynitrite � Nitric oxide � Nitrotyrosine �SLE � Histone proteins � Lysine � Arginine � Photoadduct

Abbreviations

SLE Systemic lupus erythematosus

3-NT 3-nitrotyrosine

NO Nitric oxide

O��2 Superoxide anion

RNS Reactive nitrogen species

nDNA Native DNA

RNOS Reactive nitrogen oxide species

Introduction

Systemic lupus erythematosus (SLE) is a chronic multi-

system autoimmune disease with a wide range of clinical

manifestations and pathogenesis. Its pathology is still not

fully understood, and its etiopathogenesis has remained

relatively elusive. Clinical symptoms commonly appear in

different organs such as the skin, kidney, musculoskeletal

and hematologic systems, but can also affect the lungs,

central nervous system and serous membranes [1, 2]. The

disease is responsible for significant morbidity and mor-

tality [3, 4]. Both genetic and environmental factors have

been linked to SLE [5, 6]. Environmental triggers include

R. Ahmad

Department of Biochemistry, Oman

Medical College, Sohar, Oman

H. Ahsan (&)

Department of Biochemistry, Faculty of Dentistry, Jamia Millia

Islamia (A Central University), Okhla, New Delhi 110025, India

e-mail: hahsan@rediffmail.com

123

Clin Exp Med (2014) 14:1–11

DOI 10.1007/s10238-012-0222-5

smoking [7], sunlight [8], drugs [9] and certain viruses [5].

SLE involves the innate and adaptive immunopathologies

with both endogenous and exogenous triggers contributing

in the initiation and progression of the disease such as viral

infections, ultraviolet (UV) light exposure and certain

drugs [10].

The nuclear antigens are generated and released in vivo

during apoptosis. The defective clearance of apoptotic

cellular debris in SLE patients causes a loss of self-toler-

ance, autoantibody generation and the formation of

immune complexes [11–14]. Several clinical manifesta-

tions of SLE are thought to be the result of autoantibody

and immune complex deposition in tissues leading to a

secondary inflammatory response [15]. The inflammatory

responses may involve the reaction of nitric oxide (NO)

and superoxide anions (O��2 ) leading to the formation of a

reactive nitrogen species such as peroxynitrite.

It has been established that not only oxygen but nitro-

gen-free radicals play an important role in the pathogenesis

of several human diseases. Nitric oxide radicals participate

in some pathological conditions such as arthritis, vasculitis,

asthma, hypertension, etc. It is also an unstable molecule-

like oxygen-free radical but less reactive and can react with

proteins and other biomolecules [16].

Peroxynitrite is a reactive nitrogen species, making it a

potent oxidizing and nitrating species. In vivo, it is pro-

duced by the reaction between NO and O��2 . It is formed in

the macrophages, endothelial cells, platelets, leukocytes,

neurons, etc. by the reaction between NO and O��2 . Tissue

inflammation and chronic infection lead to the overpro-

duction of NO and O��2 , which rapidly combine to yield

peroxynitrite. The studies suggest that most of the cyto-

toxicity attributed to NO is due to peroxynitrite [17].

Evidence suggests a role of reactive nitrogen species in

the initiation and progression of autoimmune phenomenon.

In chronic inflammatory diseases, peroxynitrite generation

by the phagocytic cells may cause damage to DNA and

proteins, generating neoantigenic epitopes that lead to the

production of antibodies cross-reacting with native mac-

romolecular structures. Therefore, the generation of neo-

epitopes on the molecules, may be one of the factors for the

induction of an immune response as seen in autoimmune

diseases such as SLE. Hence, understanding the contribu-

tion of peroxynitrite and other nitrogen-free radicals in the

etiopathogenesis of SLE could lead to better perception of

these systemic autoimmune disorders and the design of

new therapies directed against the disease process.

Since a lot of literature is available on the contribution

of nitric oxide in the pathogenesis of various diseases, this

review article will focus on the effects of peroxynitrite on

biomolecules (particularly amino acids, proteins and

nucleosomes) in autoimmune phenomenon especially SLE.

Peroxynitrite, a reactive nitrogen species

Peroxynitrite is a relatively long-lived oxidant that may

serve as an important cytotoxic agent. Its biological effects

are due to its reactivity toward a large number of molecules

including lipids, amino acids and nucleic acids. It is

involved in tissue damage in a number of pathophysio-

logical conditions such as neurodegenerative diseases,

cardiovascular disorders, etc. [18–20]. Peroxynitrite inter-

acts with lipids, DNA and proteins causing oxidative

damage and other free radical-induced chain reactions.

These reactions trigger cellular responses such as cellular

signaling, oxidative injury, committing cells to necrosis or

apoptosis. In vivo, peroxynitrite generation represents a

crucial pathological mechanism in conditions such as

stroke, myocardial infarction, chronic heart failure, diabe-

tes, inflammation, neurodegenerative disorders and cancer.

The cell signaling cascade that depends on reversible

phosphorylation of tyrosine is disrupted by tyrosine nitra-

tion. Thus, the occurrence of nitrotyrosine containing

proteins in vivo may be regarded as a general indicator of

tissue damage induced by peroxynitrite [21, 22]. The

nitrosative stress under certain in vivo conditions can result

in the formation of carcinogenic nitrosoamines [23–26].

Both mammalian and bacterial cells when exposed to NO

lead to deamination of guanine, cytosine and adenine via

nitrosative reactions [27].

Peroxynitrite exhibits unique chemical reactivities such

as protein nitration, DNA strand breakage, base modifica-

tion, etc., which may have cytotoxic effects and also lead to

mutagenesis. It is thought to be involved in both cell death

and an increased cancer risk [28–30]. The reaction of

peroxynitrite with lipids leads to peroxidation (malondial-

dehyde and conjugated diene formation) and formation of

nitrito-, nitro-, nitrosoperoxo- and nitrated lipid oxidation

derivatives [31–33]. Peroxynitrite is a particularly effective

oxidant of aromatic molecules, thioethers and organosulfur

compounds that include free amino acids and polypeptide

residues.

Peroxynitrite can influence protein and enzyme function

by nitration of tyrosine residues in tissues leading to

pathological malfunction [34]. The formation of 3-ni-

trotyrosine (3-NT) is contributed by many reactive nitrogen

species such as peroxynitrite, nitrogen dioxide, nitrous

acid, nitronium ion. NO alone is not capable of nitrating

tyrosine [35]. In addition, another way of tyrosine nitration

involves myeloperoxidase, secreted by monocytes and

polymorphonuclear neutrophils under inflammatory con-

ditions [36]. The nitrated product, 3-NT, is found in many

pathophysiological conditions such as chronic inflamma-

tion, atherosclerosis, acute lung injury, etc. [37, 38].

The formation of nitrotyrosine represents a specific

2 Clin Exp Med (2014) 14:1–11

123

peroxynitrite-mediated protein modification; thus, detec-

tion of nitrotyrosine in proteins is considered as a bio-

marker for endogenous peroxynitrite activity [31, 39–41].

Even though nucleic acid antigens are by themselves

poorly immunogenic, their antigenicity can be enhanced by

modification through different free radicals [20]. DNA

damage also occurs by RNOS formed under oxidative

stress. This damage is primarily caused due to formation of

peroxynitrite [42].

Autoimmunity

It has been postulated that the immune response against

host antigens could be due to genetic predisposition,

exaggerated B cell activity or cross-reactivity between

foreign and host antigens. The foreign antigens arise as a

consequence of infection, inflammation, drugs, environ-

mental factors, free radicals, etc. [43, 44].

The mechanism of autoantibody production in diseases

such as SLE has not yet been clearly identified. If antigen

selection is an important aspect of differentiation, the

nature of the stimulating antigen also remains to be

determined. The origin of antibodies remains an enigma,

although modified DNA appears to be a causative factor in

RA and SLE. It is possible that the production of autoan-

tibodies may be the result of free radical attack on chro-

matin or histone proteins causing changes at the

macromolecular level. It is, therefore, postulated that in

chronic inflammatory diseases, free radicals generated by

phagocytic cells may cause damage to DNA and proteins

and antibodies to self-antigen are produced. Also, a defect

in the control of apoptosis and delayed clearance of

apoptotic debris provides sustained interaction between

free radicals and macromolecules, generating neoepitopes

which subsequently result in autoimmunity and generating

poly-specific autoantibodies [45].

The origin of autoantibody remains ambiguous, and the

production of anti-DNA antibodies is even more intricate.

Even though nucleic acid antigens are themselves poorly

immunogenic, their antigenicity can be enhanced by

modification with agents such as free radicals. The auto-

antibodies produced against modified biomolecules are the

characteristic feature of systemic human disease, SLE. B

cell hyperactivity and the production of pathogenic auto-

antibodies are the main immunological event in the path-

ogenesis of this disease. The amino acid and nucleotide

sequence of autoantibodies derived from human lupus

subjects with active disease show features of diversification

with a high rate of replacement or silent mutations and the

clustering of mutations in the hypervariable region. This

distinctive feature implies that a pure polyclonal activation

cannot be the only mechanism responsible for autoantibody

production. An antigen-driven process is more likely to

play a role in their generation. It has been suggested that

the antibodies may be stimulated by nucleic acid antigens

or pathogens. B cells whose paratopes have complementary

determining regions (CDR) which are formed by amino

acids that can promote DNA binding may be selectively

stimulated by nucleic acid-related structures [16, 17].

Systemic lupus erythematosus

Two diseases that are considered as a prototype for sys-

temic autoimmunity are SLE and rheumatoid arthritis

(RA). SLE is a multi-systemic disorder characterized by a

variety of autoantibodies and abnormal lymphocyte func-

tion that are responsible for many of the clinical manifes-

tations important in diagnosis. A hallmark of SLE is the

presence of antinuclear antibodies (ANA). The ANA are

prototype autoantibodies that distinctly mark the course of

rheumatic diseases [46]. Because of the close association

between ANA and clinical diagnosis, these antibodies have

become a key component in the evaluation of patients.

These antibodies target a diverse range of macromolecules

including DNA, RNA, proteins and protein–nucleic acid

(PNA) complexes.

SLE is a chronic multisystem and multifactorial

inflammatory autoimmune disease, characterized by a

range of autoantibodies directed against the native nucle-

osome; its DNA and/or its histone components. Nuclear

antigens are generated and released in vivo during pro-

grammed cell death or apoptosis. A hallmark of apoptosis

is the cleavage of chromatin by caspase-activated deoxy-

ribonuclease (DNase) (CAD) [47]. CAD is an endonucle-

ase that is activated by active caspase 3 during apoptosis

and is responsible for degradation of chromatin into

nucleosomal units. The presence of apoptotic bodies is

significant for the generation of autoantigen in autoimmune

diseases, such as SLE, which is characterized by the

presence of antinuclear antibodies [48]. The initial immu-

nogen(s) that drive the development of SLE are unknown,

but characteristics of the immune response in SLE suggest

that it is an antigen-driven response, and a modified

chromatin antigen(s) could be one of the immunogens for

the production of antinuclear antibodies of various types in

SLE [49].

Other factors implicated in the development or pro-

gression of SLE include altered cytokine levels, altered sex

hormone metabolism, increased apoptosis and oxidative

stress. In oxidative stress conditions, there is an evidence of

increased levels of phospholipid peroxidation products and

plasma DNA oxidative products, 8-oxo-20-deoxyguanosine

[50].

Antibodies to DNA have been particularly associated

with SLE which is considered to be a prototype

Clin Exp Med (2014) 14:1–11 3

123

autoimmune disease. Native DNA is no longer regarded as

the antigen initiating the disease due to the fact that

immunization of experimental animals with nDNA does

not produce SLE-like symptoms. A few of the possible

candidates could be polynucleotides, denatured DNA,

RNA or modified DNA. While antibodies to single-stran-

ded DNA are formed in several inflammatory complexes

including RA; antibodies to double-stranded DNA serve as

an immunological marker in the diagnosis of SLE [51].

Serum obtained from SLE individuals has been shown to

possess anti-DNA antibodies of diverse antigenic speci-

ficity. These anti-DNA autoantibodies have been used to

evaluate therapeutic effect and clinical features of SLE

patients [52, 53].

Among protein modifications, protein tyrosine nitration

is widely recognized as a hallmark of inflammation that is

associated with the upregulation of inducible nitric oxide

synthase [54]. Both free and protein-associated 3-NT are

commonly found in mammalian tissues and are dramati-

cally increased in pathological conditions previously

associated with the production of reactive oxygen and

nitrogen species [55]. Elevated levels of RNS in SLE

patients suggest a possible role in the pathogenesis of the

disease. Murine models of SLE demonstrate abnormally

high levels of RNS compared with normal mice, whereas

systemic blockade of RNS production reduces disease

activity [56]. A correlation between serum nitrate/nitrite

(NO3/NO2) levels and serum nitrotyrosine levels with

lupus disease activity has been observed. Nitration of

tyrosine is irreversible and can be detected as long as the

nitrated protein is in circulation [57]. Subjects with active

lupus nephritis have higher levels of serum nitrotyrosine

than those without renal disease, suggesting that overpro-

duction of NO and its derived species may have a patho-

logical role in SLE and lupus nephritis [57, 58].

Elevated levels of NO in SLE patients suggest a role of

NO in the pathogenesis of the disease. Murine models of SLE

demonstrate abnormally high levels of NO compared with

normal mice, whereas systemic blockade of NO production

reduces disease activity. Elevated serum nitrate levels cor-

relate with disease activity and along with serum titers of

anti-(dsDNA) antibodies serve as an indicators of SLE [59,

60]. Auto-antibody production in SLE has been attributed to

either selective stimulation of autoreactive B cells by self-

antigens or antigens cross-reactive with self. The persistence

of anti-DNA antibodies in SLE patients, despite systems to

suppress self-recognition, suggests that the response is dri-

ven by an antigen-resembling nDNA. The DNA damage by

peroxynitrite is far more lethal than that caused by NO alone,

leading to the perturbations in nDNA that renders it immu-

nogenic. The modified DNA might, therefore, play a role in

the induction of circulating anti-DNA autoantibodies in

various autoimmune disorders including SLE [61, 62].

Anti-nDNA autoantibodies are considered a diagnostic

marker of SLE. They are expressed in association with

multiple ANA specificities, suggesting a role for both

generalized and antigen-specific immune abnormalities in

their etiology [63, 64]. Histones are small cationic proteins

that bind DNA and remain confined to the nucleus. They

are weak immunogens, probably because of their con-

served nature. However, after apoptosis, they may appear

in the circulation as nucleosomes. Histones show strong

immunogenicity after acetylation and nitration [65]. The

occurrence of autoantibodies against histone proteins has

been reported in sera of SLE patients [66]. In addition, anti-

nDNA autoantibodies are generally present with anti-his-

tone autoantibodies and may react with each of the 5

chromatin-associated histones, as well as with the H3–H4

complex [66]. It has been suggested that alteration in the

amino acid structure or sequence may generate neoepitopes

on self-proteins, leading to an autoaggressive immune

attack. Furthermore, autologous proteins may also become

immunogenic if they are structurally modified. The modi-

fications may generate or mask antigenic epitopes and

stimulate relevant B cells and/or T cells, leading to a

breakdown or bypass of tolerance.

Role of peroxynitrite in autoimmunity

A number of studies support the role of free radicals in the

initiation and progression of autoimmune response. There-

fore, in chronic inflammatory diseases, peroxynitrite gener-

ated by phagocytic cells may cause damage to DNA and

proteins, generating neoantigens that lead to the production

of antibodies cross-reactive with nDNA or proteins. Modi-

fication of native DNA or proteins by peroxynitrite might

also lead to the generation of neoepitopes on the molecule

and may be one of the factors for the induction of the immune

responses as seen in the autoimmune disease-like SLE [66,

67]. The peroxynitrite-modified human DNA was found to

be highly immunogenic in rabbits inducing high titer

immunogen-specific antibodies. The data demonstrate that

the antibodies, though cross-reactive with various nucleic

acids and polynucleotides, preferentially bind peroxynitrite-

modified epitopes on DNA [67, 68].

Immunopathology of peroxynitrite: effect on DNA

DNA is a non-immunogenic entity, but any significant

unrepaired alteration in its basic structure could turn it

‘‘foreign,’’ leading to the activation of immune pathways.

A change in the structure of DNA could either be due to

radiation or interaction with different free radicals. NO and

its derivatives are among the radicals known to interact

with DNA and are primarily involved in deamination of

4 Clin Exp Med (2014) 14:1–11

123

DNA bases. Peroxynitrite, on the other hand, leads to more

extensive damage than that caused by an equivalent dose of

NO. The formation of peroxynitrite occurs both intracel-

lularly inside macrophages and extracellularly causing

DNA strand breaks and modification of guanine [69]. The

two main products identified from the reaction of deoxy-

guanosine with peroxynitrite are 8-oxodeoxyguanine and

8-nitroguanine. The former is regarded as a reliable bio-

marker for monitoring DNA damage in with various oxi-

dizing agents. The peroxynitrite-modified DNA has been

shown to acquire immunogenicity and was suspected to be

one of the causes for generation of autoantibodies in cancer

and autoimmune disorders [61, 70].

The peroxynitrite-modified DNA is a potent immunizing

stimulus, inducing high-titer immunogen-specific antibod-

ies in rabbits. Peroxynitrite modification may generate

potential neoepitopes against which antibodies are raised.

The analysis of cross-reactivity indicates that anti-perox-

ynitrite-DNA IgG is immunogen-specific, showing differ-

ent amount of cross-reactivity attributable to sharing of

common antigenic determinants. The common antigenic

determinants between peroxynitrite-DNA and nDNA could

possibly be the sugar-phosphate backbone, since perox-

ynitrite attacks DNA and causes single-strand breaks

through sugar fragmentation. Induced antibodies also rec-

ognized synthetic polynucleotides, representing A/B con-

formations, with a preference for the B-form [61].

The studies in our laboratory have investigated the

photochemical addition of lysine and arginine to native

DNA in view of its potential importance in the photo-cross-

linking of histones to DNA in chromatin [62, 71]. The C-2

carbon atom of thymine in DNA undergoes a covalent

photoaddition reaction with the e-amino group of lysine on

UV irradiation to form a DNA-lysine photoconjugate or

photoadduct [43]. The UV spectroscopic analysis of the

DNA-lysine photoadduct showed hyperchromism, indicat-

ing either the formation of single-stranded breaks in DNA

or ‘‘breathing’’ of a double-stranded polymer at the site of

lysine conjugation. Peroxynitrite caused substantial dam-

age to the DNA-lysine adduct as evident from the hyper-

chromicity of the spectral curve, which could be attributed

to the generation of strand breaks (Fig. 1a). On peroxyni-

trite modification, the hypochromicity increased, which

may be due to the shielding effect of lysine, limiting the

sites for peroxynitrite action. Hypochromicity may also be

attributed to the extensive cross-linking between perox-

ynitrite and the DNA-lysine adduct [62]. The UV spectral

analysis of the DNA-arginine photoadduct showed changes

in DNA spectra as a result of photomodification. A

hyperchromic effect was observed due to increase in

absorbance for the DNA-arginine photoadduct when

compared with that of native DNA and the unirradi-

ated DNA-arginine complex as control (Fig. 1b).

Hyperchromicity represents the formation of single-stran-

ded regions due to adduct formation. The UV absorbance

ratios (A260/A280) of DNA-arginine adduct decreased from

that of native DNA [71]. The results of these studies have

also been summarized in Table 1 [62, 71].

Immunopathology of peroxynitrite: effect on amino

acids and proteins

The free radical-mediated damage to proteins results in the

modification of amino acid residues, cross-linking of side

chains and fragmentation. The aromatic amino acids

(tyrosine, tryptophan and phenylalanine) and cysteine are

more susceptible to modification affecting the general

properties of protein, such as change in ionic charge,

hydrophobicity and conformation [72–74]. L-tyrosine and

1 – DNA-arginine adduct 2 –native DNA3 –peroxynitrite-modified adduct4 –arginine

1

2

3

4

Wavelength (nm)

Ab

sorb

ance

0.8

0.6

0.4

0.2

0200 250 300 350 400

A

B

Fig. 1 a Effect of peroxynitrite on human DNA-lysine photoadduct.

UV absorption spectra of lysine (times symbol), native DNA

(triangle), DNA-lysine adduct (square) and peroxynitrite-modified

adduct (diamond) (Ahmad et al. [62]). b Effect of peroxynitrite on

human DNA–arginine photoadduct. UV absorption spectra of argi-

nine (times symbol), native DNA (diamond), DNA-arginine adduct

(square) and peroxynitrite-modified DNA-arginine adduct (triangle)

(Ahmad et al. [71])

Clin Exp Med (2014) 14:1–11 5

123

protein-bound tyrosine are vulnerable to attack by various

reactive nitrogen intermediates, including peroxynitrite to

form 3-NT and protein-associated 3-NT [75]. Tyrosine

nitration is a post-translational modification in proteins that

occurs under conditions of oxidative stress and plays a role

in the pathogenesis of diseases such as asthma [76], Alz-

heimer’s disease [77], atherosclerosis, endotoxic shock,

human acute lung injury [78], SLE [58] and rheumatoid

arthritis [37]. The presence of nitrated proteins in vivo

indicates that nitrated polypeptides derived from the pro-

teolytic degradation of these proteins could serve as anti-

gens. Hence, antibodies that recognize 3-NT have been

successfully raised in rabbits and mice [79, 80].

Alterations in the amino acid sequence or structure can

generate neoantigenic epitopes from self-proteins, causing

aggressive autoimmune attack. There is a range of possible

post-translational modifications of proteins (transglutami-

nation, glycosylation, oxidation, nitration and proteolytic

cleavage) that allow recognition of these self neoepitopes

activating B cells and/or T cells, thus leading to the

breakdown or evasion of immune tolerance [81, 82]. These

alterations can lead to acute and chronic disease states and

may be predictors of disease risk.

The reaction of various amino acids with peroxynitrite

leads to the following products: (a) cysteine and glutathione

are converted to disulfides; (b) methionine is converted to

sulfoxide or is fragmented to ethylene and dimethyl disulfides.

Dimethyl sulfoxide is oxidized to formaldehyde; and

(c) tyrosine and tryptophan undergo one electron oxidation to

radical cations, which are hydroxylated, nitrated and dimer-

ized [83–85]. Exposure of amino acids, peptides and proteins

to ionizing radiation such as gamma radiation and peroxyni-

trite in the presence of O2 gives rise to hydroperoxides. These

hydroperoxides decompose to oxygen- and carbon-centered

radicals on exposure to copper (Cu?) and other transition

metal ions. Hydroperoxide formation on nuclear proteins

results in oxidative damage to associated DNA. These

hydroperoxide-derived radicals react readily with nucleosides

and pyrimidine bases in DNA to form adduct, for example,

8-oxo-dG. This adduct is highly mutagenic and induces G:C to

T:A transversions in human DNA after replication [86].

Both chromatin and histone proteins are components of

the nucleosome [87]. Each nucleosome is composed of 2

molecules each of H3 and H4 histones, flanked by a dimer

of histones-H2A and H2B and surrounded by about

140–200 bp of DNA. Histone H1 is located on the outside

of the nucleosome core which helps in keeping the struc-

ture locked or ‘‘stapled’’. Antinuclear antibodies in human

and murine lupus can distinguish complex epitopes that

result from the ordered interactions between histones and

DNA [88, 89]. Monoclonal antibodies to H2A-H2B-DNA

or other more complex nucleosome epitopes have been

isolated from spontaneously autoimmune mice [90, 91].

Histones are small highly conserved cationic proteins

which bind DNA and are major components of chromatin

and remain confined to nucleus. They are poorly immu-

nogenic because of their conserved nature. However, after

apoptosis, they may appear in circulation as nucleosomes.

The incidence of autoantibodies against various histones

(H1, H2A, H2B, H3 and H4) has been reported to be

between 30 and 60 % in the sera of SLE patients [66].

Histones also act as autoantigens in humoral factors-med-

iated neoplastic diseases [92]. Furthermore, anti-histone

antibodies have also been reported in conditions such as

polymyositis/dermatomyositis [93].

As peroxynitrite reaction involves free radical interme-

diates, it may favor cross-linking and aggregation during

nitration. Formation of tyrosyl radical by peroxynitrite and

its reaction with another tyrosyl radical may generate O,O0-dityrosine covalent cross-links. Peroxynitrite induces an

array of modifications in H2A structure, for example, tyro-

sine nitration, protein carbonyl, dityrosine and crosslinking.

Such gross structural changes might favor polymerization of

native epitopes of H2A histone into potent immunogenic

neoepitopes. The histone proteins are conserved proteins and

act as weak immunogens. However, they show enhanced

immunogenicity after acetylation and alterations in amino

acid structure or sequence and can generate neoantigens on

self-proteins causing and immune attack. The oxidative and

nitrative action of peroxynitrite confers additional immu-

nogenicity on H2A histone indicating a direct correlation

between nitration and immunogenicity. Hence, immuniza-

tion with peroxynityrite-modified H2A may produce poly-

specific antibodies which can recognize and bind to both old

and newly generated epitopes [45].

Contribution of peroxynitrite in SLE

SLE is a complex autoimmune inflammatory disease, and

people are affected by this disorder worldwide. Despite

Table 1 UV absorption and

thermal melting characteristics

of native and modified lysine

and arginine photoadducts

(Ahmad et al. [62, 71])

Parameter/property DNA–lysine

photoadduct

DNA–arginine

photoadduct

Peroxynitrite-modified

DNA-lysine adduct

Peroxynitrite-modified

DNA-arginine adduct

A260/A280 1.2 1.1 1.0 1.1

Tm (oC) 70 61 85 69.5

% Hyperchromicity 52 66 84 69

6 Clin Exp Med (2014) 14:1–11

123

decades of intensive research, the origin of SLE has not

been traced. Although the presence of anti-native DNA

autoantibodies characterizes SLE, but native DNA has

been rejected as an antigen-initiating SLE. A previous

report on binding of SLE anti-dsDNA antibodies to a

peptide DNA surrogate tilts the support in favor of protein

antigens as likely initiators of SLE [94]. Cells of the

immunoregulatory network produce both NO and O��2during oxidative burst triggered by inflammation which

may combine to generate peroxynitrite. The peroxynitrite-

driven oxidation and nitration of biomolecules (protein,

lipid, DNA) may lead to SLE-like autoimmunity and age-

related diseases [95, 96].

Furthermore, nitration of tyrosine residues can pro-

foundly alter protein function, suggesting that protein

nitration may be fundamentally related to, and be predic-

tive of, oxidative cell injury. The subsequent release of

altered proteins may enable them to act as antigen-inducing

antibodies against self-proteins. The biological significance

of tyrosine nitration is of interest, because evidence sup-

ports the formation of 3-NT in vivo in diverse pathologic

conditions and 3-NT is thought to be a relatively specific

marker of oxidative damage mediated by peroxynitrite.

Free/protein-bound tyrosine are attacked by various RNS,

including peroxynitrite, to form free/protein-bound 3-NT,

which may provide insight into the mechanism of severe

disease activity seen in lupus patients and among those

developing nephritis [97]. In addition, numerous other

disease states using non-human models have been shown to

involve the formation of 3-NT [55]. At the very least, the

presence of 3-NT in biological samples indicates that

reactive NO-derived species were produced in vivo,

although the exact nature of these species remains to be

determined. The biological markers that have been impli-

cated in the etiopathogenesis of autoimmune phenomenon

and/or produced as a result of reactive nitrogen species

modified-molecules have been identified and include his-

tone proteins, collagen, various types of DNA structures

and modified amino acids. Table 2 lists the various bio-

logical molecules modified by nitric oxide (2a) and per-

oxynitrite (2b) involved in the pathogenesis of autoimmune

conditions such as SLE, RA and OA [38, 56, 58, 98–104].

Sera of animals immunized with native and perox-

ynitrite-modified histones were tested on polysorp wells

coated with respective immunogens as studied by Khan

et al. [45] (Fig. 2). Treatment with 100 lm peroxynitrite

Table 2 Role of reactive nitrogen species (a) nitric oxide, (b) peroxynitrite-modified biomolecules in various pathological (autoimmune)

conditions

Molecular target Probable mechanism Pathological indication References

(a)

Calf thymus DNA Generation of neo-antigen epitopes SLE [98]

N-hydroxy-L-arginine Increased nitric oxide synthase activity RA, SLE [99]

Plasmid DNA Generation of neo-antigen epitopes SLE [98]

Poly-L-tyrosine Generation of 3-NT RA, OA [38]

Poly-L-tyrosine Generation of 3-NT SLE [100]

Serum proteins Generation of 3-NT SLE [58]

Synovial tissue proteins Generation of nitrated (nitrotyrosine) proteins RA, OA [101]

(b)

Histone protein Generation of nitrotyrosine, dityrosine, carbonyls SLE [102]

Histone protein Generation of 3-NT RA [103]

Placental DNA Generation of neo-antigen epitopes SLE [56]

Type II collagen Generation of neo-antigen epitopes RA [104]

SLE systemic lupus erythematosus, RA rheumatoid arthritis, OA osteoarthritis

Fig. 2 Antigenicity of peroxynitrite-modified histone protein. Direct

binding enzyme-linked immunosorbent assay (ELISA) of experimen-

tally induced antibodies against native histone H2A (filled square)

and peroxynitrite-modified H2A (open square) (Khan et al. [45])

Clin Exp Med (2014) 14:1–11 7

123

conferred a higher immunogenicity on H2A histone than

other lower concentration of peroxynitrite tested [45]. It

is speculated that anti-histone antibodies observed in

autoimmune conditions such as SLE patients might

originate from immunological activity of peroxynitrite-

modified histones. Peroxynitrite-modified H2A histone

could act as an autoantigen leading to generation of anti-

H2A histone antibodies. It is anticipated that anti-histone

antibodies seen in a sub-group of SLE patients might

originate from immunological activity of peroxynitrite-

modified histones due to their protection from digestion

by normal proteolytic machinery [44]. In the context of

anti-histone antibodies in drug-induced lupus erythema-

tosus, it is quite possible that the drug itself might mimic

reactions or pathways leading to abnormal synthesis of

peroxynitrite. The peroxynitrite may then modify the

structure of histone making it immunogenic [17].

As reported by Dixit et al. [102], the tested SLE sera

showed higher binding with peroxynitrite-modified H2A

histone compared with native DNA or native H2A histone

(Fig. 3). These observations are important in view of the

immunogenic nature of peroxynitrite-modified H2A his-

tone and the reported incidence of hyper-nitrotyrosinemia

and anti-histone antibodies in some SLE patients [102].

Conclusion

It has now been established clearly that not only free rad-

icals play an important role in the pathogenesis of several

human diseases. Reactive nitrogen species is produced by

the reaction of NO with O��2 or peroxide. NO participates in

some many pathological conditions such as arthritis,

autoimmune diseases, vasculitis, asthma, hypertension, etc.

As mentioned earlier, peroxynitrite is a short-lived

species that is produced by the reaction of nitric oxide

(NO) and superoxide radicals (O��2 ). Peroxynitrite is more

reactive than NO and diffuses rapidly across biological

membranes to attack various biomolecules in cells and

cellular compartments [39] resulting in their modification

[105]. Furthermore, apoptotic defects and impaired

removal of apoptotic cells contribute to an overall load of

autoantigens [106].

The amino acids and proteins may be oxidized and/or

nitrated by peroxynitrite leading to tyrosine nitration,

dimerization and hydroxylation and formation of 3-ni-

trotyrosine, dityrosine and dihydrophenylalanine, respec-

tively [107]. A strong correlation between tyrosine

nitration and inactivation of protein function has been

established for numerous proteins in different diseases [39–

41, 108]. The formation of nitrotyrosine represents a spe-

cific peroxynitrite-mediated protein modification; thus,

detection of nitrotyrosine in proteins is considered as a

biomarker for endogenous peroxynitrite activity.

A poor understanding of the pathogenesis of SLE has

hampered the development of new therapies directed at the

underlying disease process. Hence, alteration of proteins

resulting from photomodification or peroxynitrite could

lead to the development of antibodies. Therefore, the basic

amino acid (lysine, arginine)-modified proteins or modified

photoadducts could have important implications in carci-

nogenesis and autoimmunity (Fig. 4). Therefore, under-

standing the pathophysiology of peroxynitrite and nitric

oxide-modified biomolecules could lead to a better

Fig. 3 Direct binding ELISA of serum from SLE patients. Enzyme-

linked immunosorbent assay of serum from SLE patients binding with

native DNA (gray bar), native H2A (white bar with dot) and

peroxynitrite-modified H2A (white bar). Black bars correspond to

control human serum binding with the respective antigens (Dixit et al.

[102])

ProteinsPeroxynitrite

Protein modification (tyrosine nitration, crosslinking, protein carbonyl, etc)

Peroxynitrite -modified biomarkers (amino acids, histones)

Apoptotic pathway, ubiquitin-proteasomal clearance

Generation of polyclonal antibodies

no immune response

Systemic autoimmunity (SLE, RA)

Fig. 4 Peroxynitrite-mediated damage to macromolecules leading to

pathophysiological changes and conditions. Peroxynitrite may dam-

age proteins and other biological molecules leading to various

pathophysiological conditions including autoimmune phenomenon

such as SLE, etc.

8 Clin Exp Med (2014) 14:1–11

123

understanding of the autoimmune phenomenon in diseases

such as SLE.

Acknowledgments RA is grateful to the administration of Oman

Medical College, Sohar Campus, Oman for the continued support

during the preparation of this manuscript. We express our sincere

regrets if citations of some authors may have been inadvertently

missed in the review article.

Conflict of interest None.

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