Keywords
osteoporosis - tumor necrosis factor-alpha - receptors, tumor necrosis factor
Introduction
Osteoporosis is a systemic disease characterized by decreased bone mass with deterioration
of the skeletal microarchitecture, leading to bone fragility and increasing the propensity
to fractures.[1]
Bone metabolism is active throughout life, and renewal is a constant process in which
osteoclast pulls out the mineral component and osteoblast resets it.[2] The normal skeletal maturation process involves the accumulation of bone mass up
to about 30 years of age. From then on, there is a physiological loss of about 0.3%
each year, initiating the osteopenia process, with decreased bone mineral density,
weakening, and increased bone trabeculations. Osteoporosis can affect both men and
women, and may occur in any region, with the main bones being the ones in the hip
joint, the wrist, the spine, and the ribs. The weakening caused by low bone density
is related to most low-impact fractures in the elderly.[3]
Several components of the innate and adaptive immune response have been related to
the modulation of osteoclast and osteoblast activity, thus leading to direct changes
in the bone matrix.[4] Several signaling pathways were identified as contributors to the interaction between
osteoblasts and osteoclasts, including RANK, NF-Kb receptor activator, and its ligand
(RANK-L).[5]
[6]
[7]
The connection of RANK-L to its receptor, RANK, provides the signal to conduct the
development of osteoclasts from hematopoietic progenitor cells, in addition to activating
mature osteoclasts. Osteoprotegerin, also known as an osteoclastogenesis inhibitor,
is the TNF-alpha receptor-related protein (alpha tumor necrosis factor) that controls
the development and function of osteoclasts. It is responsible for the negative regulation
between RANK-L and its receptor, therefore inhibits bone resorption by osteoclasts.[8]
[9]
TNF-alpha is a potent cytokine, which exerts a variety of biological effects, and
can perform a pleiotrophic role in the immune response, inflammation, in addition
to controlling cell proliferation, differentiation and apoptosis.[10]
[11] Tumor necrosis factor alpha acts on osteoclastogenesis through a mechanism involving
the activation of NF-kB.[12]
[13] In addition, TNF-alpha acts by directly stimulating macrophages to differentiate
into osteoclasts by an independent mechanism of RANK-L.[14]
[15]
Currently, it is known that TNF-alpha binds to 2 cell surface receptors, type 1 receptor
(TNF-R1), also known as p55, and type 2 receptor (TNF-R2) or p75 sections, each receptor
being responsible for mediating distinct intracellular signals.[16]
Both TNF-R1 and TNF-R2 are highly expressed in osteoclast precursors.[17] When activated by TNF-alpha, the type 1 receptor stimulates osteoclastogenesis by
activating NF-Kb and inhibits the osteoblasts differentiation.[18]
[19] On the other hand, type 2 receptor activation showed osteoclastogenesis suppression
in in vitro experiments.[17]
The present study aims to evaluate the levels of TNF-alpha and its receptors, TNF-R1
and TNF-R2, in elderly patients with low-impact fractures due to osteoporosis, comparing
and analyzing the values between men and women and with patients in the control group.
Materials and Methods
All procedures of the present study were approved by the Research Ethics Committee
of the University under protocol (CAAE) number: 51827515.4.0000.5145 and opinion number
1,375,317, and all participants signed a Free and Informed Consent Form, after clarification.
Peripheral blood samples were collected from 62 patients, 21 women and 13 men, with
low-impact fractures due to osteoporosis, and 11 women and 17 men who presented high-impact
trauma fractures, forming patients in the control group ([Table 1]).
Table 1
|
Group
|
Number
|
Average age (years old)
|
Standard error
|
p-value
|
|
Control woman
|
11
|
40.9
|
3.1
|
0.83
|
|
Control man
|
17
|
42.6
|
3.2
|
|
|
Osteoporosis woman
|
21
|
80.1
|
2.1
|
0.14
|
|
Osteoporosis man
|
13
|
78.9
|
2.6
|
|
The collection was conducted in the orthopedic center of our institution. Patients
with bone diseases, fractures not related to osteoporosis, immunosuppressed patients,
patients with malignant neoplasms and those who did not agree to participate in the
study were excluded.
ELISA For Measuring Sly Cytokines
Tumor necrosis factor alpha (TNF-alpha) and tumor necrosis factor receptor types 1
and 2 (TNFR1 and TNFR2) were evaluated by enzyme immunoabsorption assay (ELISA). Plates
of 96 high-affinity bonding wells (Nunc, Roskilde, Denmark) were sensitized with monoclonal
antibodies specific to each cytokine investigated (BD Biosciences, San Jose, CA, USA)
under the conditions recommended by the carbonate-bicarbonate buffer manufacturer
(pH 9.5) and were incubated overnight at 4°C. After incubation, the contents of the
wells were discarded and the plates were blocked with 200 μL per phosphate buffer
well containing 2% buffered saline solution with bovine serum phosphate/albumin (PBS-BSA)
(Sigma, St. Louis, MO, USA) for 4 hours at room temperature. Bovine serum phosphate/albumin
was then discarded and lines 1 to 10 were filled with diluted serum samples 1:2 in
1% of PBS-BSA for a total volume of 100 μL per well. Serial dilutions of recombinant
cytokines were used to elaborate the standard curve. The plates were incubated overnight
at 4°C and then rinsed 4 times with PBS solution containing Tween at 0.05% (Sigma,
St. Louis, MO, USA). Next, biotin-conjugated antibodies (BD Biosciences, San Jose,
CA, USA) specific to each cytokine investigated were added to wells diluted with PBS-BSA
at 1% at the concentration indicated by the manufacturer for a total of 100 μL per
well. The plates were incubated for 4 hours at 37°C, rinsed with 0.05% PBS Tween,
treated with 100 μL per strepavidin well conjugated with peroxidase and then incubated
for 3 hours at 37°C. Finally, the plates were rinsed with 0.05% PBS-Tween. A total
of 100 μL of tetramethylbenzidine (TMB) development buffer was added per well under
light and at room temperature; the reaction was interrupted by the addition of 50
μL of sulfuric acid. The results were obtained by measuring absorbance at 450 nm using
an automatic ELISA reader (Enspire, Perkin Elmer, USA). Cytokine concentrations were
determined based on linear regression using absorbance values obtained from recombinant
cytokine curves and are expressed in pg/ml. The sensitivity of the method ranged from
10 to 18 pg/ml.
Statistical analysis
Statistical analysis was performed in GraphPad Prisma 7.0 and Statview (Abaccus, USA)
software. The normality of quantitative variables was investigated using the Kolmogorov-Smirnov
test and the Shapiro-Wilk test. The Student t-test was used to analyze clinical parameters
such as age. Cytokine levels were analyzed by the Mann-Whitney test. The significance
level was established at 5% (p < 0.05) in all quantitative tests.
Results
Tumor necrosis factor alpha serum levels were undetectable in all female patients.
In the male group, it was detected in only one patient. The median was zero in both
groups, with no significant difference (Mann-Whitney; p = 0.38) (data not shown).
Tumor necrosis factor-R1 serum levels ranged from 1,209 to 4,918 pg/ml (median of
2,720 pg/ml) in the group of women with osteoporosis. In men with osteoporosis, levels
ranged from 1,257 to 4,273 pg/ml (median of 2,680 pg/ml). There was no significant
difference between the groups of patients with osteoporosis when analyzed by gender
(Mann-Whitney; p = 0.74). In the control group, TNF-R1 serum levels ranged from 369
to 2,000 pg/ml (median of 689 pg/ml) in the group of women. In men, levels ranged
from 198 to 2,417 pg/ml (median of 608 pg/ml). Tumor necrosis factor-R1 levels showed
a significant increase in the osteoporosis group compared with the control group in
both females and males (p < 0.0001) ([Fig. 1]).
Fig. 1 Comparison of TNF-R1 serum levels between study groups in patients of both sexes.
Tumor necrosis factor-R2 serum levels had similar results, ranging from 1,313 to 5,042
pg/ml (median of 2,495 pg/ml) in the group of women with osteoporosis. In men with
osteoporosis, the levels ranged from 1,276 to 4,271 pg/ml (median of 2,618 pg/ml).
There was no significant difference between the groups of patients with osteoporosis
when analyzed by gender (Mann-Whitney; p = 0.85). In the control group, TNF-R2 serum levels ranged from undetectable to 2,023
pg/ml (median of 1,350 pg/ml) in the group of women. In men, the levels ranged from
744 to 5,753 pg/ml (median of 1,380 pg/ml). Tumor necrosis factor-R2 levels showed
a significant increase in the osteoporosis group compared with the control group in
both females and males (p < 0.0001 and 0.033, respectively) ([Fig. 2]).
Fig. 2 Comparison of TNF-R2 serum levels between study groups in patients of both sexes.
As a significant increase in TNF-R1 and TNF-R2 was observed in men and women with
osteoporosis, we tested whether there would be a correlation between TNF-R1 and TNF-R2
serum levels in osteoporosis, both in males and females. The results presented in
[Figs. 3] and [4] show that there is a positive and significant correlation only in females (Sperman;
p = 0.049).
Fig. 3 Correlation between TNF-R1 and TNF-R2 serum levels in women with osteoporosis.
Fig. 4 Correlation between TNF-R1 and TNF-R2 serum levels in men with osteoporosis.
Discussion
Osteoporosis develops from an imbalance in bone remodeling, which results from both
hormonal changes and immunological factors. In the present study, we analyzed the
presence of TNF-alpha, a potent cytokine in the inflammatory role, and its receptors,
TNF-R1 and TNF-R2, in patients with osteoporosis and in the control group, also comparing
between genders.
The results showed significant differences in the amount of TNF-R1 and TNF-R2 receptors.
Women with osteoporosis have high levels of TNF-R1 and TNF-R2 receptors when compared
with women in the control group. Similarly, men with osteoporosis have high levels
of TNF-R1 and TNF-R2 receptors when compared with men in the control group.
Studies show that the increase in proinflammatory cytokines, such as TNF-alpha, may
be directly related to aging, and inappropriate production of this cytokine or sustained
activation of its signaling pathways are factors related to the development of osteoporosis.[20]
[21] However, as well as the present study, other authors have demonstrated no association
between changes in TNF-alpha levels between normal women and men and those with osteoporosis.[22]
[23]
In the present study, we also evaluated the serum levels of TNF-alpha receptors. The
evaluation of soluble forms of TNF-alpha receptors should be evaluated,[24] since the dosage of circulating levels of TNF-alpha based on bone mass status is
inconsistent and controversial.[22]
[23] Studies show that the activation of TNF-R1 leads to increased osteoclastogenesis
and decreased differentiation of osteoblasts.[21]
In the present study, serum levels of TNF-alpha, TNF-R1, and TNF-R2 receptors were
analyzed. We found a significant increase when comparing TNF-R1 levels in women and
men with osteoporosis with those in the control group, corroborating that there is
a relationship between the physiopathogenesis of osteoporosis and the effects of TNF-R1
activation. Similarly, we showed a significant increase in TNF-R2 serum levels in
patients with osteoporosis, both in the male and female groups. The activation of
TNF-R2 has been described as suppressive of osteoclastogenesis.[25] Our data show that TNF-alpha was not detected in the serum of patients with osteoporosis
and that serum levels of their TNF-R1 and TNF-R2 receptors are increased when compared
with the control group. Signaling of these receptors is associated with modulation
of osteogenesis and, if stimulated inadequately, it may result in bone homeostasis
imbalance as demonstrated in studies that observed imbalance in bone deposition, in
the occurrence of weakened differentiations of osteoblasts and adipocytes.[26]
An interesting finding was the fact that there is a positive and significant correlation
between the levels of TNF-R1 and TNF-R2 only in women, suggesting that the two receptors
are released homogeneously only in females.
Conclusion
The present study demonstrates a significant increase in TNF-alpha, TNF-R1, and TNF-R2
receptors in patients with osteoporosis in men and women. In females, serum levels
of these receptors present a significant and positive correlation, while this correlation
was not observed in males. These data suggest that the expression and release of these
receptors may be contributing differently to the development of osteoporosis in men
and women.
