Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160
*Corresponding author:
Douglas Wright, PhD
Department of Anatomy and Cell Biology
University of Kansas Medical Center
Kansas City, KS 66160 Tel: 913-588-2713 Fax: 913-588-2710 E-mail: dwright@kumc.edu
Received November 11, 2011; Accepted December 17, 2011; Published December 22, 2011
Citation:Wilson NM, Wright DE (2011) Inflammatory Mediators in Diabetic
Neuropathy. J Diabetes Metab S5:004. doi:10.4172/2155-6156.S5-004
Diabetic neuropathy (DN) is the most common complication of diabetes mellitus, resulting in high health care
costs and greatly affecting the quality of life of patients. The mechanism(s) responsible for the development of DN
is currently unclear, but is likely multifactoral. While a great deal is still unknown about the mediators responsible for
the development of DN, current knowledge of other neuropathic conditions suggest the involvement of a number of
inflammatory mediators such as interleukins and chemokines. This review will outline the current literature of a number
of inflammatory mediators in both human and rodent studies. As there is currently no therapeutic treatment for DN, this
review will also provide an overview of therapeutics targeting inflammation and the ability of these therapies to improve
the symptoms of DN.
Introduction
Diabetes mellitus currently affects 25.8 million individuals in the
United States with total costs in the US reaching $174 billion (www.
diabetes.org). Diabetes is associated with a number of complications
including: heart disease, stroke, blindness, kidney disease, and
neuropathy. Diabetic neuropathy (DN) is the most common
complication of diabetes, with 60-70% of diabetic patients reporting
mild to severe forms of peripheral nerve dysfunction (www.diabetes.org). DN is characterized by a number of neural symptoms including
numbness, sensory loss, and stabbing or burning pain typically
experienced in the hands and feet. These sensory symptoms are thought
to associate with the progressive loss or damage to sensory nerve
fibers. A number of mechanisms for DN have been proposed, ranging
from increased reactive oxygen species production, increased protein
glycosylation, neurovascular disturbances, and decreased neurotrophic
support [1]. One area that has had comparatively limited investigation
is the role of inflammatory mediators in DN. Inflammatory mediators
such as cytokines have been linked to many forms of neuropathic pain.
Thus, the role that cytokines play in DN should merit exploration and
will be the focus of this review.
Nuclear Factor Kappa B (NF-κB)
NF-κB is a transcriptional factor that is activated by a number
of stimuli and is responsible for initiating the transcription of a
number of different inflammatory and immune mediators. Therefore,
monitoring the activation of this transcription factor can be used as an
indicator of the state of the inflammation in tissue. Analyzing various
tissues from diabetic patients has revealed increased NF-κB activity
in the kidney, endothelial cells, peripheral blood mononuclear cells,
monocytes, and sural nerve [2-5]. Further characterization has been
conducted in rodent studies from which NF-κB has been identified as
a factor involved in models of diabetic neuropathy. NF-κB activation
is increased in the dorsal root ganglion (DRG), sural, and sciatic nerve
of diabetic mice compared to non-diabetic mice [3,6]. Investigations
into the mechanism of increased NF-κB activation have demonstrated
that NF-κB activation could be prevented in receptor for advanced
glycation end products (RAGE) null mutant mice [6]. The involvement
of glycosylated proteins and RAGE has been explored in a number of
complications of diabetes, but whether RAGE signaling also leads to
inflammatory changes needs further exploration.
Toll-Like Receptors
Toll like receptors (TLR) are pattern recognition receptors utilized by the innate immune system. Activation of TLRs induces NF-κB
activation and increases the expression of a number of cytokines and
chemokines. Two of the most highly characterized toll-like receptors,
TLR2 and TLR4, are expressed in a number of cell types including
monocytes. High glucose treatment of human monocytic cell lines
increases the expression of TLR2 and TLR4 in a dose-dependent
manner. Additionally, high glucose treatment of these cells also
leads to increased release of IL-1, IL-6, MCP-1, and TNF-α, which is
prevented in TLR2 and TLR4 knockdown conditions [7]. Follow-up
studies utilizing monocytes from human patients further confirmed
the in vitro findings. Monocyte TLR2 and TLR4 surface expression and
mRNA levels were significantly increased from Type 1 Diabetic patients
compared to healthy controls [5]. This group went on to demonstrate
significantly higher circulating levels of TLR2 and TLR4 ligands, highmobility
group box 1 (HMGB1) and heat shock protein 60 (Hsp60), in
Type 1 patients compared to control patients [8].
While changes in TLRs have been demonstrated in diabetic
patients, the link between those changes and DN is just beginning to
be explored. Two common single nucleotide polymorphisms exist
in the TLR4 gene at Asp299Gly and Thr399Ile. The presence of these
polymorphisms alters the structure of the extracellular domain, which
has the potential to alter ligand binding [9]. Analysis of the TLR4
gene in diabetic patients revealed that the presence of Asp299Gly and
Thr399Ile genotypes reduced the incidence of DN in Type 2 patients.
While the TLR4 polymorphisms also occurred in Type 1 patients
the protection against the development of DN was not witnessed
[10]. However, the number of Type 1 patients in the study was lower
than Type 2 patients which might alter the outcome. The possible
mechanism behind the protection of these polymorphisms against
the development of DN could stem from changing the extracellular
structure of TLR4 and potentially altering the ligand binding domain.Combining this data with the presence of increased TLR4 ligands could
explain this protection, by preventing the increased inflammatory
response induced by TLR4 signaling.
Cytokines
Interleukins
Interleukins (IL) are a group of cytokines named for their ability to
communicate between leukocytes. In recent years, research has shown
their involvement in cell signaling in a number of other cell types
and tissues. There are more than 30 interleukin isoforms currently
identified that can have either pro-inflammatory (IL-1,IL-6 and IL-8)
or anti-inflammatory (IL-4 and IL-10) actions. Interleukins have been
shown to be involved in a number of different neuropathic conditions
in both animal and human studies. Clinical studies in small-fiber
neuropathy patients demonstrated a two-fold increase in circulating
IL-2 mRNA levels in peripheral blood compared to healthy controls.
Skin samples from the affected area were collected from patients, which
showed increased IL-6 and IL-8 mRNA levels compared to controls.
Further analysis comparing the affected skin area to unaffected skin
areas in this patient population revealed increases in IL-1β, IL-6, and
IL-8 mRNA levels, 2-fold, greater than 200 fold, and greater than
500 fold, respectively [11]. While significant research has been done
in other neuropathic conditions, research on the role interleukins
play in diabetic neuropathy is just beginning to be explored. Rodent
studies utilizing streptozotocin (STZ) injections to induce DN have
revealed increased in IL-6 mRNA levels in both the DRG and sciatic
nerve compared to non-diabetic mice [3,12]. Clinical findings utilizing
sural nerve biopsies from DN patients demonstrated increased IL-6
expression compared to healthy controls [3]. Expansion of these
findings with particular focus on how interleukins contribute to the
symptoms of DN such as nerve conduction velocity and fiber loss is
still needed.
Chemokines
Chemotactic cytokines, or chemokines, are an integral part of the
immune system and in recent years have been shown to be involved in
nociceptive behavior. The role of chemokines in nociceptive behavior
was been demonstrated through hindpaw injections of chemokines,
stromal derived factor 1 (SDF1) and regulated on activation normally
T-cell expressed and secreted (RANTES), inducing decreases in
mechanical threshold [13]. The chemokines role in neuropathic
pain states has also been shown in both drug- and injury- induced
neuropathy rodent models, such as anti-HIV treatment and chronic
constriction injury [14]. Chemokines role in diabetic neuropathy
has begun to be explored as well. Rodent studies have demonstrated
monocyte chemoattractant protein- 1 (MCP-1) is increased in both
plasma and adipose tissue in diabetic ob/ob mice compared to nondiabetic
lean mice [15]. Clinical studies utilizing serum samples from
diabetic patients have demonstrated significant increases in MCP-1
and RANTES levels compared to controls [16,17]. Guan and colleagues
expanded on their findings by revealing a significant correlation
between increases in serum MCP-1 levels and diabetic complications,
including peripheral neuropathy [17]. The link between DN and
chemokines was strengthened by reports of significantly increased
levels of circulating RANTES in DN patients when compared to
healthy controls and diabetic patients with no symptoms of neuropathy
[18]. While the association between chemokines and DN is correlative
at this time, given the demonstrated role chemokines play in other
neuropathies, further exploration is warranted, including changes in
chemokine expression within the peripheral nervous system.
Tumor Necrosis Factor α (TNF-α)
TNF-α is a pro-inflammatory cytokine that has been implicated in
neuropathic and inflammatory nociceptive conditions for a number
of years. This link stems from the ability of TNF-α administration
to induce ectopic firing in sensory neurons [19] and mechanical and
thermal nociceptive behavior [20] in rodents. More confirmation of
the link between TNF-α and nociceptive behavior is evidenced by the
fact that TNF-α mRNA expression corresponds to the time course of
the development of thermal and mechanical behavior in a neuropathic
pain model using chronic constriction injury [21].
Recently TNF-α has also begun to be implicated in diabetic
neuropathy. Clinical studies have shown increased TNF-α plasma
protein and mRNA levels in diabetic patients compared to controls
[22-24]. Increased TNF-α macrophage expression and plasma levels
were also demonstrated in DN patients compared to controls and was
correlated to pain intensity [22]. Exploration of these clinical findings
in rodent models has been key to understanding its role in DN. STZinduced
DN in rodents results in increased circulating levels of TNF-α
[25,26,27,28] and the consequence of increased TNF-α expression
was explored in STZ-injected rats. TNF-α administration into the
sciatic nerve induced a reduction in motor nerve conduction velocity
(MNCV), a symptom commonly witnessed in patients with DN [29].
TNF-α’s role in the symptoms of DN has been validated in studies
utilizing TNF-α null mutant mice. TNF-α deficient diabetic mice
fail to develop changes in nociceptive behavior, MNCV, and sensory
nerve conduction velocity (SNCV) compared to diabetic mice with
wild type TNF-α expression [12]. To further characterize TNF-α role
in diabetic neuropathy, neutralization studies were conducted using
the TNF-α neutralizing anti-body, infliximab. Infliximab treatment
following STZ injections recovered MNCV and SNCV losses, tail flick
nociceptive behavior, and prevented a loss of epidermal nerve fibers
compared to STZ-injected control animals [12]. Treatment with the
TNF-α neutralizing antibody was also effective in reducing circulating
TNF-α serum levels and TNF-α mRNA expression in the DRG back
to control animal levels [12]. Thus, these knockout and neutralizing
studies highlight a likely important role of TNF-α in the development
of DN.
While, TNF-α stands out as a cytokine with the most evidence
to support a role for cytokines in DN, research also suggests that
chemokines and interleukins could also be involved. Moving forward,
studies expanding on the correlative studies and moving towards a
more mechanistic approach are needed.
Cyclooxygenase
Cyclooxygenase (COX) is the rate-limiting enzyme for
prostaglandin synthesis and its inhibition is the target of most nonsteroidal
anti-inflammatory drugs (NSAIDs). There are two COX
isoforms identified as COX-1 and COX-2. COX-1 is constitutively
expressed, while COX-2 has limited expression under normal
conditions, but is induced in inflammatory conditions. The inducible
nature of COX-2 makes it a good candidate for monitoring the
inflammatory state of tissue. Diabetic rodent studies have revealed
altered cyclooxygenase expression, including increased COX-2 protein
levels in the sciatic nerve and sensory neurons of STZ injected rodents
[30,31]. Insight into the role of COX-2 in the development of DN was
explored using COX-2 deficient mice. These studies suggested that the
absence of COX-2 protected STZ-injected animals against decreases
in SNCV, MNCV, endoneurial blood flow, and intraepidermal nerve
fiber density compared to nondiabetic mice [31,32]. The prevention of these characteristic DN symptoms supports a critical role COX-2 in
DN. Importantly, the absence of COX-2 expression in COX-2 deficient
diabetic mice was effective at mediating these effects on nerve function
for up to 6 months following STZ injections [31]. These studies suggest
an ongoing role for COX enzymatic activity in the development of DN.
Therapeutic Treatments Targeting Inflammatory
Mediators in Diabetic Neuropathy
With the discovery that inflammatory mediators such as TNF-α are
increased in DN, researchers began to focus on therapeutic treatments
that could target these inflammatory mediators. In vitro experiments
using monocytes from type 2 diabetic patients demonstrated increased
expression of TNF-α, IL-1, IL-6, and IL-8 compared to healthy controls
and type 1 diabetic patients. Treatment of human monocytes from type
2 patients with the active form of vitamin D, 1,25-dihydroxyvitamin
D3, downregulates the mRNA expression of TNF-α, IL-1, IL-6, and
IL-8 [33]. While these in vitro experiments demonstrated the ability
of therapeutics to target inflammatory mediators in DN, further in
vivo experiments were needed. In vivo rodent experiments using the
natural flavonoid, curcumin, dose-dependently decreased serum
TNF-α levels and attenuated thermal hyperalgesia in STZ-treated mice
[28,34]. The beneficial effect of curcumin treatment was enhanced with
co-treatment with insulin [28]. Additional therapeutics capable of
preventing inflammatory mediated events in rodent models included
gliclazide, a sulfonylurea used in non-insulin dependent diabetes, and
troglitazone, a thiazolidinedione. Both gliclazide and troglitazone
attenuated TNF-α levels and improved MNCV in STZ-injected rats.
These treatments also prevented decreases in myelinated fiber area,
fiber density, and axon/myelin ratio in the tibial nerve of diabetic rats
[25,26]. An additional therapeutic that was tested was the anti-oxidant,
N-acetylcysteine, which dose dependently improved TNF-α levels and
MNCV in STZ-induced diabetic rats [27]. While all the mentioned
therapeutics do not belong to a unifying drug class, they were all shown
to have an effect on TNF-α levels and were able to help in preventing
the development of DN.
NSAIDS are a classical therapeutic for inflammatory conditions
and are available in both over-the-counter and prescription forms. As
stated previously, this therapeutics’ mechanism of action is targeted at
inhibiting the enzyme COX. NSAIDS include both non-selective COX
inhibitors such as ibuprofen and selective COX-2 inhibitors such as
celecoxib. Animal studies exploring the therapeutic potential of these
compounds in DN have begun to be explored following the discovery
of increased COX-2 levels in peripheral tissues in DN models. Nonselective
COX inhibitors have shown mixed results. Piroxicam was
shown to statistically increase STZ-induced decreases in sensory
neuron action potential amplitude [35]. The non-selective inhibitors,
sulindac and indomethacin, corrected losses in sural and caudal sensory
nerve conduction velocity of diabetic rodents compared to control
mice [36,37]. While some non-selective COX inhibitors have shown to
be an effective treatment option, flurbiprofen treatment alone induced
decreases in MNCV. Thus, flurbiprofen treatment mimicked STZinduced
changes and lacked the ability to alter STZ-induced changes
on MNCV [30]. The authors suggested inhibiting the ubiquitously
expressed COX-1 is responsible for these effects, indicating COX-1
may be responsible for maintaining neural function in rodents [30].
Given this observation, the use of selective COX-2 inhibitors was
utilized to determine their effectiveness. Celecoxib treatment prevented
MNCV and SNCV slowing [31], and meloxicam treatment was shown
to protect against MNCV slowing and endoneurial blood flow deficits
in diabetic rodents [30]. Further characterization through intrathecal administration of COX-2 inhibitors revealed a dose dependent
attenuation of mechanical behavior [38]. Another interesting effect
of selective inhibition of COX-2 through pharmacological or gene
inactivation is a prevention of increased TNF-α expression in the
sciatic nerve of STZ-induced diabetic rodents [31].
Limited corresponding research has been done in clinical studies.
However, one patient study has been conducted evaluating NSAID
treatment in diabetic patients, which demonstrated an improvement
of neuropathy score with ibruprofen and sulindac treatment compared
to placebo treatment of the same patients [39]. The interpretation of
these results is limited because of the lack of a healthy age-matched
control group. Comparisons in this study were only made between
responders and non-responders among the DN patients. While
NSAID treatment is a promising avenue to explore with a number
of currently available treatment options, known side effects of this
class of drugs would be prohibitive to long-term treatment. Although
selective COX-2 inhibitors would prevent against gastrointestinal
side effects, the cardiovascular effects of COX-2 inhibitors could be
problematic, especially in a patient population already at increased risk
for cardiovascular complications.
Therapeutic treatments aimed at targeting inflammatory mediators
in DN thus far have shown effects when the therapeutic is administered
at the time of DN induction. There is a lack of evidence to demonstrate
their effectiveness after the development of DN in reversing any of the
symptoms of DN such as reductions in nerve conduction velocities or
nociceptive behavior. It is highly unlikely the initiation of therapeutic
treatment would coincide with the initial development of diabetes
in patients. Therefore, studies investigating the time course of antiinflammatory
therapeutics are needed. While current studies haven’t
addressed reversal of DN, a notable finding from a limited number of
studies is that treatment effects were ineffective in initial time points,
but instead the statistically significant beneficial effects were only
evident after 12 weeks of treatment [25,26]. This temporal aspect may
suggest the inflammatory component of DN might not develop until
later time points. Therefore, administration of therapeutics at later time
points might still be effective, but further studies are needed to validate
these findings.
Cytokine Expression Profile in Painful versus Painless
Neuropathy
One interesting component of DN is the presence of both painful
and painless presentation in human and rodent studies. The cause
of this presentation of both painful and painless neuropathies is
unclear. One theory put forth by Uceyler and colleagues points toward
differences in the cytokine expression profile. Their study evaluated
both pro- and anti-inflammatory cytokine levels in painful and painless
patient populations. The patient population for the study encompassed
a number of neuropathic pain conditions, including diabetic
neuropathy and chronic inflammatory demyelinating neuropathy.
The authors were able to demonstrate that patients with painful
neuropathy had 2-fold greater levels of TNF-α and IL-2 compared to
controls and painless neuropathy patients. Another notable difference
in the cytokine profile of this patient population was found when
analyzing the painless patients samples which had statistically higher
circulating levels of the anti-inflammatory cytokine, IL-10, twofold
higher compared to controls [24]. These findings are interesting
given the recent publication from Saleh and colleagues that showed a
decrease in TNF-α levels in the DRG of STZ-injected rats [40]. These
findings are in stark contrast to other papers that have demonstrated an increase in TNF-α levels in patients and diabetic rodent models [22-
28]. One reason for this discrepancy could be attributed to differences
in the phenotypic expression of the nociceptive behavior in different
models of DN. For example, previous studies have found diabetic
rodents with decreased DRG TNF-α levels demonstrate hypoalgesic
behavior [40], while diabetic rodents with increased DRG TNF-α levels
display hyperalgesic behavior [12]. Whether this is consistent with all
rodent DN models is unclear. However, support for increased TNF-α
levels contributing to nociceptive behavior has been demonstrated
in previous publications independent of a DN state [20,21]. These
results would suggest that individuals with painful neuropathies
have a predominantly pro-inflammatory cytokine expression profile,
while patients with painless neuropathies are more susceptible to antinflammatory
cytokine expression. In all likelihood, the predisposing
factor that makes a patient susceptible to develop a painful versus
painless neuropathic pain state is likely from differences in genetic
make-up, but the mechanism responsible for these differences is
unknown.
There are other possible confounding factors that could be
contributing to the differences in cytokine levels in painful and
painless neuropathy patients. For example, depression, which has
also been shown to correspond with increased cytokine levels, could
be contributing the differences witnessed. Uceyler and colleagues
acknowledge in their conclusion that the painful neuropathy
patients had higher depression scores than painless patients [24].
This assumption is made stronger with the evidence that higher
TNF-α levels were present in depressed painful neuropathy patients
compared to those without depression [41]. Whether depression is the
determining factor for differences in cytokine levels, or if the decreases
in quality of life resulting from increased pain itself are contributing to the depression is unclear. Another variable associated with the time
of sample collection and duration of illness needs to be addressed.
This variable was suggested by Empl and colleagues as contributing
to the results of their study that found increased Schwann cell TNF-α
expression in painful neuropathy patients compared to non-painful
patients [42]. The authors noted that the painful neuropathy patients
in their study had a shorter duration of illness, explaining this by the
painful symptoms prompting patients to seek medical attention earlier.
The validity of this concern stems from inflammatory mediators, such
as TNF-α, are often reported in the early stages of nerve injury, during
time periods when the immune system is responding to the insult.
Taking this into account, the initial immune response would be early
and probably provide a minor contribution to when the samples were
collected. In fact, there is no significant difference in the duration
of illness in the painful and painless neuropathy patients from the
previously cited study (76.14 ±24.17 months vs. 94.99±21.58 months),
and the duration of the illness would be well past an initial immune
response [24]. Taking into account the outlined confounding variables
that could be contributing, follow-up studies with a greater number of
patients, particularly DN patients, and with samples from a number of
time points would further validate these findings.
Conclusions
The increase of inflammatory mediators in a number of
neuropathic pain states has been well documented. The potential role
of these mediators in DN is just beginning to be explored, with TNF-α
and COX-2 currently demonstrating the strongest involvement (for
summary of current results refer to Table 1). Further investigation
into additional inflammatory mediators such as interleukins and
chemokines is warranted, including rodent studies to explore the mechanisms by which these proteins could result in a number of
DN symptoms, including nerve conduction velocity and epidermal
innervation deficits. Further translational studies are needed to confirm
findings in animal models, especially expanding on the therapeutic
potential of targeting inflammatory mediators. Additionally, follow-up
studies determining whether differences in cytokine profile expression
continue when comparing painful versus painless diabetic neuropathy
patients is an interesting aspect to explore. If similar results are
evident in DN patients, it could be beneficial in determining enhanced
personalized treatments for diabetic neuropathy patients.
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