TH1 and TH2 Cytokines Production and NK Cell Level Assessment in Peripheral Blood of Patients with DDH

Abstract

In this study, our aim is; if the studies will quide us in peripheral blood, for the changes in inflammatory cytokine levels we defined before DDH tissue. Twenty-six patients were suggestive of lumbar DDH were included in this study. Control subjects included 14 autopsy cases. From each patient, disc tissues and peripheral blood samples for plasma were collected during the surgery. For the controls, disc samples and blood for plasma by intracardiac puncture were obtained during autopsy. The Flow Cytometry was used to obtain the lymphocyte CD56 (NK). The Luminex was used to obtain IL-2, IL-4, IL-10, IL-12, IFN-gamma, in both plasma and disc tissues. The results were compared between the two groups. Comparing the two groups regarding plasma demonstrated that IL–2, IL–4, IL–12, IFN-gamma were significantly higher than in patients than those of the controls. Likewise, tissue levels of IL–2, IL–4, IL–10, IL–12, TNF-alpha, CD56 were found to be significantly higher in the patients. With respect to the comparison between the plasma disc samples in the patients, plasma showed significant higher levels of IL-2, IL-12 on the other hand IL-4 was found to be significantly higher in the disc samples. Findings suggest that only tissue samples responses in occurring but not blood samples. We don’t think our results in peripheral blood will guide us specifically in DDH.

Introduction

Degenerative disc disease (DDD) is a common cause of pain and disability, characterized by anatomic, morphologic, and biochemical changes, including altered expression of both matrix metalloproteinases and proinflammatory cytokines [1]. Elevated levels of molecular mediators of inflammation have been described in pathologic disc tissue, increasing with the grade of degeneration [2, 3].

These symptoms are caused by the fact that worn-out discs are a source of pain because they do not function as well as they once did, and as they shrink, the space available for the nerve roots also shrinks. As the discs between the intervertebral bodies start to wear out, the entire lumbar spine becomes less flexible [4, 5].

Nonspecific degeneration is termed ‘DDH’ (developmental dysplasia of the hip) or ‘spondylosis’. As the patient ages, the proteoglycan content of the disk nucleus decreases, tears develop on the disk annulus, fibrous tissue develops in the disc, and anatomic changes such as height loss occur. However, the mechanisms by which neural inflammation and pain occur have not been fully elucidated [4, 5].

Intervertebral disc (IVD) degeneration starts with serious pain, inflammation, and local nerve irritation, later resulting in disc herniation. The inflammatory and immune activation profile exhibited by a herniated disc may be distinct from that observed in the IVD degeneration and may differ further among different extents of herniation from protrusion through sequestration. Histologic evaluation of herniated disc tissue has revealed prominent infiltration of inflammatory cells, most markedly macrophages [6–11]. Various inflammatory cells, proinflammatory cytokines, antibodies, and immunoglobulins have been identified in and around herniated lumbar disc tissue. Recently, it has been reported that lumbar disc tissue may be another potential immune-privileged site in the human body [6–11].

In DDD, various degrees of hyperinflammatory activity follow periods of compensatory hypoinflammation and periods of immunosuppression. During these periods abnormalities may be observed such as diminished phagocytosis and intracellular killing activity, diminished monocyte/macrophage activity and in T and B lymphocytes, reduced immunoglobin synthesis changed and cytokine levels [12]. Although the immune system is thought to form a powerful response to these degenerations, details of these mechanisms do not appear in the literature. We observed any relative changes between the major proinflammatory cytokines (TH1) (IL-1β, IL-2, TNF-α, IFN-γ) and the anti-inflammatory cytokines (TH2) (IL-10, IL-4) and to observe the effects of any such changes on NK (Natural Killer) and any resultant interactions with IL-12 [12].

In this study, our aim is; if the studies will guide us in peripheral blood, for the changes in inflammatory cytokine levels we defined before DDH tissue.

Material and Method

This work was conducted at the Neurosurgery and Physiology Departments of the Cerrahpasa Faculty of Istanbul University and at the Medical Institute of the Turkish Ministry of Justice. In 26 patients aged 47.46 ± 11.25 years, 16 males (61 %) and 10 females (39 %), who had applied to our neurosurgery clinic complaining of sciatica and found by magnetic resonance imaging (MRI) to have lumbar DDT and acute radiculopathy, surgery was indicated. This surgery was conducted with their informed written consent.

Control subjects included 14 autopsy cases. From each patient, peripheral blood samples for plasma were collected during the surgery. For the controls, blood for plasma by intracardiac puncture was obtained during autopsy. The following cell levels were also obtained by flow cytometry: NK(CD56) which plays the major defense role in the innate immune system, CD3, CD4, CD8 for the cellular system and for the humoral system, the main cell CD19(B lymphocyte) along with CD40 and CD25. The multiple analyte platform (XMAP, Luminex) was used to obtain IL-1β, IL-2, IL-4, IL-10, IL-12, IFN-γ, and TNF-α in plasma. The results were statistically compared between the two groups.

Statistical Analysis

All data collected from each patient were organized in a database (Excel, Microsoft Corp.). Numeric variables were provided as the mean ± SD. For statistical analysis, we used the nonparametric Mann–Whitney U test and chi-square test for categorical variables. For correlation analysis, the Pearson correlation was used and a probability value of less than 0.05 was considered statistically significant. All statistical calculations were performed using commercially available software (SPSS version 12.0, SPSS Inc.).

Results

We showed all the results in Tables 1 and 2.

Table 1.

A summary of statistical results showing the comparisons regarding the major cytokines and lymphocytes in the blood sample here between the two groups

Plasma	Control	Patientss	P
Ages	37,21 ± 14,9	47,46 ± 11.25	0,019
Male/Female	10 (%71) / 4 (%29)	16 (%61,5) / 10 (%38,5)	0,047
IL–1 B(pg/ml)	6,42 ± 2,05	46,6 ± 29,03	0,0001
IL–2(pg/ml)	7,62 ± 2,61	26,11 ± 6,39	0,0001
IL–4(pg/ml)	17,15 ± 3,44	11,24 ± 2,99	0,0001
IL–10(pg/ml)	23,96 ± 13,62	27,55 ± 11,28	0,378
IL–12(pg/ml)	7,74 ± 1,53	9,13 ± 2,16	0,040
IFN- γ (pg/ml)	4,62 ± 2,19	8,94 ± 3,36	0,0001
TNF-α (pg/ml)	18,87 ± 7,08	20,55 ± 4,33	0,044
CD3(%)	24,13 ± 12,41	88,76 ± 7,3	0,0001
CD4(%)	15,97 ± 5,99	64,23 ± 14,24	0,0001
CD8(%)	8,89 ± 5,74	21,61 ± 8,04	0,0001
CD56(%)	4,07 ± 2,51	2,86 ± 1,78	0,798
CD25(%)	1,40 ± 1,36	1,57 ± 0,52	0,491
CD19(%)	3,05 ± 2,17	1,57 ± 0,57	0,0001
CD40(%)	0,6 ± 0,14	7,96 ± 4,01	0,0001

Significant differences (p < 0.05)

Table 2.

Summary of statistical results showing the comparisons regarding the major cytokines and lymphocytes in the disc samples and plasma studied here in patient groups

Parameters	Tissue	Plasma	P
IL–1 β(pg/ml)	31,61 ± 7,82	29,03 ± 8,36	0,228
IL–2(pg/ml)	11,81 ± 2,51	26,11 ± 6,39	0,0001
IL–4(pg/ml)	32,18 ± 10,38	11,24 ± 2,99	0,0001
IL–10(pg/ml)	30,67 ± 9,57	27,55 ± 11,28	0,258
IL–12(pg/ml)	7,33 ± 2,15	9,13 ± 2,16	0,002
IFN- γ (pg/ml)	8,16 ± 3,95	8,94 ± 3,36	0,446
TNF-α(pg/ml)	28,94 ± 9,04	20,55 ± 4,33	0,0001
CD3(%)	25,76 ± 6,88	88,76 ±7,30	0,0001
CD4(%)	20,34 ± 6,26	64,23 ±14,24	0,0001
CD8(%)	5,53 ± 2,15	21,61 ±8,04	0,0001
CD56(%)	3,50 ± 1,94	2,86 ± 1,78	0,161
CD25(%)	20,92 ± 5,87	1,57 ± 0,52	0,0001
CD19(%)	11,00 ± 6,86	9,92 ±5,25	0,474
CD40(%)	8,57 ± 5,83	7,96 ± 4,01	0,970

Significant differences (p < 0.05)

Table 1 summarizes the statistical results between the two groups. Patients showed significantly higher levels with respect to the basic cytokines and immune cells from the blood sample IL-1β, IL-2, IL-10, IFN-γ, CD3, CD4, CD8, and CD40 compared to the controls (P < 0.05). However, no significant differences were found regarding TNF-α and CD25. In the blood sample of patients, IL-4, CD56, and CD19 levels decreased significantly.

We determined that IL-1β, IL-10, IL-12, IFN-γ, CD19, CD56, and CD40 in disc samples of patients showed no significant differences compared with blood sample of patients (Table 2). Significantly higher levels in the disc samples of patients in relation to IL-2, CD8, CD3, and CD4 were found compared with the blood sample of patients. However, CD25, IL-4, and TNF-α significantly decreased (Table 2).

Discussion

Recent research shows two subgroups of T cells secreting different cytokine profiles after stimulation. On the one hand, Th1-type cells secrete high levels of IL-2, TNF-α, and IFN-γ which in turn activate macrophages and promote cell-mediated immune responses against invasive intracellular pathogens. Th2-type cells produce a variety of anti-inflammatory cytokines, including IL-4, IL-5, IL-6, IL-10, and IL-13 [13].

In our study, when DDH blood samples were compared with the cadaver control group, we determined that IL-1β and IL-2 had increased but there was no significant difference in TNF-α. In our former study, different results were obtained in DDH tissue samples from blood samples. We observed that IL-1β, TNF-α, and IL-2 had significantly increased in the disc samples of patients [12].

IL-1β, TNF-α, IL-2, and CD40 increased significantly in the blood samples of the patients. IL-1β and TNF-α provide both humoral and cellular responses. IL-2 expressed by active cells has been reported to increase IL-2 and IFN-γ production. Our findings described that IL-2 and IFN-α levels were found to be higher in patients than in controls, CD25 (IL-2R) level was no significant differences. Increasing IL-2 levels activated CD56 and CD19 cells. TNF-α affects phagocytes and stimulates anti-inflammatory cytokines such as IL-4 and IL-10 and it keeps balance among the cytokines [14].

In our study, when DDH blood samples were compared with cadaver control groups, it was determined that IL-4 had decreased and IL-10 had increased. IL-4 and IL-10 are inhibitors for Th1 cytokines (IL-2, IFN-γ, TNF-α). It should be noted that increase in IL-10 levels could play an inhibitory role on the feedback and TNF-α. IL-4 stimulates progress of Th2 cells which are from CD4 cells. It suppresses the cell-mediated immune response by an antagonist effect to IFN-γ macrophage activator effect. Although IL-10 has protective effects, increased levels are related to poor prognosis. IL-10 shows its protective effects by inhibiting IL-1β, IL-8, IL-6, IFN-γ, and TNF-α.

It can result in a significant increase in IL-1β, IL-2, and IFN-γ by suppressed IL-4 and in no significant increase in IL-10 levels of DDH in blood samples. Significant increased IL-2 and IFN-γ could have inhibitor effect on IL-10 and IL-4 which are Th2 cytokines.

IL-2 is a growth factor for CD4 cells and also activates CD56 and CD19 cells. While IL-2 stimulates antibody creation on B cells, it can stimulate CD56 to increase and increases cytolytic functions at the same time. It also alerts CD56 activation constituting synergy with IL-12 and IL-2. It is being claimed that Th2 cytokine expression has precedence in disc cells.

When blood samples of DDH were compared with cadaver control groups, there was no significant change. In IL-12 and CD56 (NK) but IFN-γ increased significantly. We had pointed out in our former study that only IL-12 increased significantly in DDH tissue. In immune-privileged disc hernia tissue, inflammation was occurring by immune reaction break cascade.

Our former study had resulted in DDH increase of IL-4 and IL-10, but in this study, we obtained cytokines (IFN-γ, IL-2), which are inhibitors for Th2 in peripheral blood as a response. This situation shows it affects privileged formation of lumbar DDH tissue and peripheral blood in different functions [12].

Results of this study differ from the former study we had done in DDH tissue, and it is important from the point of showing immune changes in peripheral blood [12]. We know that there are a lot of factors affecting peripheral blood. But despite all these factors, we suggest that a total evaluation of the immune system will be useful to define medical treatment for DDH.

Results

In the former study, we had mentioned that infections could be prevented using inhibitor cytokine blockage for Th1 and Th2 in degenerative disc space and that precautionary measures could be taken for systemic immune suppression through local use. But we do not think our results in peripheral blood will guide us specifically in DDH.