Suppressive Effects of Imidapril on Th1- and Th2-Related Chemokines in Monocytes

Abstract

Background

Angiotensin-converting enzyme inhibitors (ACEIs) are used to control hypertension and are superior to other antihypertensive agents in protecting the progressive deterioration of autoimmune-related nephritis. An imbalance of T helper 1 (Th1)/Th2 is thought to contribute to the pathogenesis of autoimmune diseases and their related glomerulonephritis. I-309 is a Th2-related chemokine involved in the recruitment of Th2 cells toward Th2-related inflammation. Tumor necrosis factor α (TNF-α) and Th1-related chemokines, interferon-inducible protein 10 (IP-10)/CXCL10 are also involved in autoimmune glomerulonephritis. However, the modulatory effects and the mechanisms of ACEIs on TNF-α and Th1- and Th2-related chemokines in monocytes remain poorly defined.

Objective

We investigated the effects of imidapril and perindopril, 2 ACEIs, on the expression of IP-10, I-309, and TNF-α in human monocytes and also the associated intracellular mechanism.

Results

Imidapril and perindopril significantly downregulated lipopolysaccharide (LPS)-induced TNF-α, I-309, and IP-10 in THP-1 cells and human primary monocytes. All 3 mitogen-activated protein kinase inhibitors suppressed LPS-induced TNF-α and I-309 expression in human primary monocytes. Only extracellular signal-regulated kinases and c-Jun N-terminal kinases (JNK) mitogen-activated protein kinase inhibitors suppressed LPS-induced IP-10 expression. Lipopolysaccharide-induced mitogen-activated protein kinase kinase 4 (MKK4), p-JNK, and c-Jun expression in human primary monocytes was suppressed by imidapril.

Conclusions

These data demonstrate that ACEI is effective in downregulating LPS-induced TNF-α, I-309, and IP-10, which play important roles in the pathogenesis of inflammation. Its suppressive effect on TNF-α, I-309, and IP-10 may, at least in part, involve the down-regulation of LPS-induced MKK4-JNK-c-Jun expression.

Keywords

imidapril TNF-α IP-10 I-309 monocyte JNK

An imbalance of T helper 1 (Th1)/Th2 is thought to contribute to the pathogenesis of autoimmune diseases and important for autoimmune glomerulonephritis (GN).^1,2 A recent report showed that T cells producing interferon γ (IFN-γ; Th1 cells) increased in patients with class IV lupus nephritis according to the World Health Organization. The serum level of monocyte chemotactic protein-1 and interferon-inducible protein 10 (IP-10; Th1-related chemokine) in patients with proliferative lupus nephritis is significantly elevated.³ The combination of Th1 inducers and chemokine inhibition might be a powerful therapeutic approach in proliferative lupus nephritis.³ Some medications for GN have been also focused on modulating the Th1/Th2 imbalance.² The role of the renin-angiotensin system in hypertension and end-organ damage has been recognized for a long time. Angiotensin I-converting enzyme inhibitor (ACEI) is superior to other antihypertensive agents in protecting progressive deterioration of renal function, even in normotensive persons. Angiotensin-converting enzyme inhibitors were the first to be used to block the renin-angiotensin system and now have many compelling indications in the treatment of hypertension and its cardiovascular and renal complications. In patients and animals with nephropathies, ACEIs improve renal function more than that would be expected from their control of hypertension.⁴ Like ACEI, angiotensin II type 1 receptor antagonists ameliorate or even reverse glomerulosclerosis in rat animal models.⁴ These findings suggest that angiotensin II has nonhemodynamic effects and anti-inflammatory effect in progressive renal disease.⁴ The ACEI/angiotensin II type 1 receptor blockade is also reported to be of benefit in blood pressure control and long-term renal survival in lupus nephritis on the basis of immunosuppressive drugs and steroids to control systemic lupus activity⁵ and also prolong graft survival in patients with post-transplant GN.⁶ However, the detailed immune modulatory effect and their related mechanism of ACEI are still not clear.

Tumor necrosis factor α (TNF-α) is not just a proinflammatory cytokine and also proposed to be an immunoregulatory molecule that can alter the balance of T regulatory cells.⁷ Polarization with cytokines influences every aspect of the immune response from the innate to the adaptive. For monocytes, cytokines influence the expression of chemokines, the expression of costimulatory molecules, and the execution of effector programs. Th1 and Th2 polarization with IFN-γ and interleukin 4 (IL-4) are 2 well-studied systems.⁸ The Th1-derived cytokine, IFN-γ, drives monocytes to an M1 phenotype, whereas the Th2 cytokine, IL-4, drives the alternative pathway of activation resulting in an M2a phenotype.⁹ Chemokines are a family of small (8-10 kd), secreted, usually inducible proteins for leukocyte recruitment, leukocyte homeostasis, and inflammatory responses.¹⁰ Fu et al.¹¹ reported that the combined transcription level of interferon-inducible chemokines in peripheral blood leukocytes is associated with disease activity, degree of organ damage, and specific autoantibody patterns in systemic lupus erythematosus (SLE). Therefore, chemokine score may serve as a new biomarker for active and severe disease in SLE. The chemokine receptor CXCR3 (the ligand of IP-10) is highly expressed on Th1-polarized T cells and has been predicted to play an important role in T-cell recruitment and immune response in a number of inflammatory and autoimmune diseases. CXCR3-deficient mice with nephrotoxic nephritis showed an increased renal messenger RNA expression of Th1-related chemokines during the autologous phase. This increased chemokine expression was also associated with the renal infiltration of T cells, followed by renal tissue injury, albuminuria, and loss of renal function.¹² Interstitial CXCR3, as well as CCR5-positive T cells might play an important role during progressive loss of renal function in patients with immunoglobulin A nephropathy and are potential therapeutic targets in human glomerular diseases.¹³ Th2-related chemokines also play some roles in SLE. The Th2-type CC chemokine thymus and activation-regulated chemokine (TARC/CCL17) and I-309 are the high-affinity ligands for CCR4, a chemokine receptor predominantly expressed by Th2 cells. TARC/CCL17 may be a useful serological marker and may facilitate an assessment of the degree of disease activity in SLE.¹⁴ The development of SLE is closely related to the elevation of plasma TARC/CCL17 levels.¹⁴

Because Th1/Th2 transcriptional regulation is important for autoimmune GN and ACEI ameliorates or even reverses glomerulosclerosis in rat animal models, it is reasonable to evaluate the effect of ACEI on the proinflammatory cytokine and Th1- and Th2-related chemokines in monocytes. We investigated whether 2 ACEIs could modulate the Toll-like receptor-mediated TNF-α, I-309 (Th2-related chemokine), and IP-10 (Th1-related chemokine) expression in monocytes and explored the detailed mechanisms of intracellular pathway, including mitogen-activated protein kinase (MAPK) and nuclear factor κB.

Methods

Cell Preparation

The human monocytic cell line THP-1 (American Type Culture Collection, Rockville, MD) was cultured in RPMI 1640 medium (Sigma Chemical, Co, St Louis, MO) supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37°C and 5% CO₂ in a humidified incubator. Cells were centrifuged and resuspended in fresh medium in 24-well plates at a concentration of 10⁶/mL for 24 hours before experimental use. The cells were pretreated with imidapril or perindopril (Sigma) 2 hours before lipopolysaccharide (LPS, 0.2 μg/mL) (Escherichia coli; Sigma) stimulation. Cell supernatant was collected 24 hours after LPS stimulation.

The study of human subjects was approved by the institutional review board of Kaohsiung Medical University, Taiwan. After obtaining informed consent, peripheral blood samples were from healthy individuals who had no personal or family history of allergies (n = 3). Peripheral blood mononuclear cells were isolated by density-gradient centrifugation (Lymphoprep, Oslo, Norway). Human primary monocytes were isolated from peripheral blood mononuclear cells by magnetic bead sorting with anti-CD14 monoclonal antibody (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany).

Enzyme-Linked Immunosorbent Assay

The TNF-α, I-309, IP-10, IL-4, IL-10, TARC, MIP-1α, MCP-1, and RANTES concentrations of cell supernatants were determined using commercially available enzyme-linked immunosorbent assay (ELISA)-based assay systems (R&D System, Minneapolis, MN). Assays were performed using the protocols recommended by the manufacturer. The lowest detection limit of the IP-10 ELISA was 31.25 pg/mL.

Mitogen-Activated Protein Kinase Assay

After 1 hour of treatment with or without 3 MAPK inhibitors (PD98059, SB203580, and SP600125, 10 μM for each), the cells were stimulated with LPS (0.2 μg/mL). The cell supernatant was collected 24 hours after LPS stimulation for the measurement of IP-10 and I-309 and TNF-α by using ELISA.

Western Blotting and c-Jun MAPK Activity Assay

After treatment for 2 hours with or without imidapril, the cells were stimulated with LPS (0.2 μg/mL) and lysed with equal volumes of ice-cold 150-μL lysis buffer 1 hour later. After centrifugation at 13,000g for 15 minutes, equal amounts of cell lysates were analyzed by Western blot with anti-p65, anti-phospho-p65, anti-MAPK (p38, extracellular signal-regulated kinases [ERK], and c-Jun N-terminal kinases [JNK]), antiphospho-MAPK (pp38, p-ERK, and p-JNK), anti-mitogen-activated protein kinase kinase 4 (MKK4), and anti-phospho-MKK4 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). JNK and c-Jun MAP kinase activities in cells were measured by nonradioactive JNK and c-Jun MAPK assay kits (Cell Signaling Technology, Danvers, MA). Assays were performed using the protocols recommended by the manufacturer. The c-Jun was used as substrates for JNK MAPK assay in vitro, and c-Jun phosphorylation was detected by Western blot using phospho-c-Jun antibody to determine JNK MAPK activity, respectively.

Statistical Analyses

All data are presented as mean (SD). Differences between experimental and control groups were analyzed by using the Mann-Whitney test. P < 0.05 was considered indicative of significant between-group differences.

Results

Imidapril and Perindopril Suppressed LPS-Induced TNF-α Expression in THP-1 Cells and Human Primary Monocytes

Imidapril (0.1 μM) significantly downregulated LPS-induced TNF-α production in THP-1 cells after LPS stimulation for 24 hours (Fig. 1A) and imidapril (0.01-0.1 μM) also significantly suppress LPS-induced TNF-α production in human primary monocytes (Fig. 1B). Next we used Toll-like 3 receptor agonist poly I:C to evaluate whether imidapril had similar effect. Imidapril (0.1 μM) did reduce the poly I:C-induced TNF-α production in human primary monocytes (Fig. 1C) after 24 hours of poly I:C stimulation. Another ACEI, perindopril (0.1 μM), also significantly reduced LPS-induced TNF-α expression in human primary monocytes (Fig. 1D). These data suggested that ACEIs might suppress different Toll-like receptor agonist-induced TNF-α production in human monocytes.

Figure 1

Imidapril suppressed the LPS-induced TNF-α production in THP-1 cells (A) and also in human primary monocytes (B) after 24 hours of LPS stimulation. Imidapril reduced the poly I:C-induced TNF-α production in human primary monocytes (C). Perindopril suppressed the LPS-induced TNF-α production in human primary monocytes (D). *P < 0.05 between LPS/poly I:C only and LPS/poly I:C plus imidapril or perindopril treatment.

Imidapril Suppressed LPS-Induced IP-10 Expression in THP-1 Cells and Human Primary Monocytes

Imidapril could not significantly downregulated LPS-induced IP-10 production in THP-1 cells (Fig. 2A). However, imidapril (0.01 μM) significantly downregulated LPS-induced IP-10 production in human primary monocytes 24 hours after LPS stimulation (Fig. 2B). Imidapril (0.1 μM) significantly suppressed poly I:C-induced IP-10 production in THP-1 cells and human primary monocytes 24 hours after poly I:C stimulation (Fig. 2, C and D).

Figure 2

Imidapril downregulated LPS-induced IP-10 production in THP-1 cells (A) and human primary monocytes (B) 24 hours after LPS stimulation. Imidapril significantly suppressed poly I:C-induced IP-10 production in THP-1 cells and human primary monocytes 24 hours after poly I:C stimulation (C and D). *P < 0.05 between LPS/poly only and LPS/poly plus imidapril treatment.

Imidapril and Perindopril Suppressed LPS-Induced I-309 Expression in Human Primary Monocytes

Imidapril (0.01-0.1 μM) significantly downregulated LPS and poly I:C-induced I-309 production in human primary monocytes (Fig. 3, A and B). We further test whether another ACEI had a similar effect. Perindopril (0.1 μM) also significantly reduced LPS-induced I-309 expression in human primary monocytes (Fig. 3C). However, perindopril (0.001-1 μM) did not suppress poly I:C-induced I-309 production in human primary monocytes (Fig. 3D) after 24 hours of LPS stimulation. These data suggested that different ACEIs might have differently suppressive effects on different Toll-like receptor agonist-induced I-309 production in human primary monocytes.

Figure 3

Imidapril significantly suppressed LPS and poly I:C-induced I-309 production in human primary monocytes (A and B). Perindopril also significantly reduced LPS-induced I-309 expression in human primary monocytes (C). However, Perindopril did not suppress poly I:C-induced I-309 production in human primary monocytes (D) after 24 hours of LPS stimulation. *P < 0.05 between LPS/poly only and LPS/poly plus imidapril or perindopril treatment.

Imidapril and Perindopril Had No Effect on LPS-Induced IL-4, IL-10, TARC, MIP-1α, MCP-1, and RANTES Expression in THP-1 Cells and Human Primary Monocytes

To further evaluate the effect of ACEI on other Th2 immunity, Th2-related cytokine IL-4 and IL-10 were also investigated. However, imidapril and perindopril had no effect on the expression of IL-4, IL-10, TARC, MIP-1α, MCP-1, and RANTES expression at 24- and 48-hour time points in THP-1 cells and human primary monocytes (data not shown).

Imidapril Suppressed LPS-Induced TNF-α, IP-10, and I-309 Expression via MKK4-JNK-c-JUN Pathway in Human Primary Monocytes

Figure 4 (A, B) shows that SB203580 (p38-MAPK inhibitor), SP600125 (JNK-MAPK inhibitor), and PD98059 (ERK-MAPK inhibitor) suppressed LPS-induced TNF-α and I-309 expression, suggesting that all 3 MAPK signals are important factors of LPS-induced TNF-α and I-309 expression in human primary monocytes. Only SB203580 and SP600125, but not PD98059, could only suppress LPS-induced IP-10 (Fig. 4C). Imidapril suppressed LPS-induced p-JNK, but not pp38, pERK, or pp65, on human primary monocytes. Therefore, the suppressive effect of imidapril on LPS-induced TNF-α, IP-10, and I-309 expression in human primary monocytes may be through the JNK MAPK pathway. MKK4 and c-Jun is the upstream and downstream signaling of JNK, respectively.¹⁵ Therefore, we next evaluated the upstream and downstream pathway of JNK. The results showed that imidapril could also decrease p-MKK4 and p-c-Jun expression (Fig. 5, A and B). These data suggested that imidapril suppressed LPS-induced TNF-α, IP-10, and I-309 expression, at least, in part through the MKK4-JNK-c-Jun pathway.

Figure 4

PD98059, SB203580, and SP600125 suppressed LPS-induced TNF-α (A) and I-309 (B) expression. SB203580 and SP600125 reduced LPS-induced IP-10 expression (C). Imidapril decreased LPS-induced p-JNK expression in human primary monocytes (D). *P < 0.05.

Figure 5

Imidapril could downregulate LPS-induced p-MKK4 expression (A). Imidapril could also decrease p-c-Jun expression (B).

Discussion

Hypertension is a common problem associated with SLE and nephritis induced by this autoimmune disorder. Systemic lupus erythematosus is an inflammatory multisystem disease of unknown etiology with immunologic aberrations, for example, imbalance of Th1/Th2. Angiotensin-converting enzyme inhibitors affect various immune phenomena through the renin-angiotensin and kallikrein-kininogen systems by creating angiotensin II and inactivating bradykinin. The ACEI being superior to other antihypertensive agents in preventing progressive deterioration of renal function suggests the involvement of ACEI on immunologic regulation. Genetic factors, including ACE gene polymorphism, are implicated in the development of SLE.¹⁶ Interestingly, in vivo or in vitro exposure to captopril reduced splenic levels of type 2 cytokines, interleukin IL-4 and IL-10, suggesting a possible role of the immune system in captopril-mediated disease modulation. Because Th2 cytokines are known to promote lupus glomerulosclerosis, decreased IL-4 and IL-10 production in captopril-treated mice may be related to this agent's renoprotective effects.¹⁷ In the present study, ACEIs not only act as proinflammatory cytokine TNF-α inhibitors but also as immunomodulators to suppress Th1- and Th2-related chemokines’ expression in monocytes. These results may explain why ACEIs are superior to other antihypertensive agents in protecting the kidney against progressive deterioration.

Angiotensin II-stimulated human endothelial cells increased the release of a CXC chemokine, IP-10, according to an antibody array. The expression of IP-10 was higher in the endothelium of coronary blood vessels in mice infused with angiotensin II than in control. Quantitative real-time polymerase chain reaction analysis revealed that angiotensin II significantly increased IP-10 messenger RNA expression compared with control.¹⁸ Interferon-inducible protein 10 is involved not only in leukocyte-endothelial interaction but also in the circuit of endothelial renin-angiotensin system activation that potentially promotes atherosclerosis.¹⁸ Therefore, the suppressive effect of ACEIs may reduce the production of IP-10 and further suppresses endothelial renin-angiotensin system activation.

In the present study, ACEIs seemed to be potent in suppressing the TLR-related production of Th1- and also Th2-associated chemokines in THP-1 cells. We further look at the intracellular signal pathway to further explore the detailed mechanisms. Mitogen-activated protein kinases and nuclear factor κB may be involved in LPS-induced chemokines’ and cytokines’ expression of monocytes.^19–21 The suppressive effect of PD95059, SB203580, and SP600125 on the expression of LPS-induced I-309 and TNF-α in human monocytes suggested that the activation of ERK, p38, and JNK may be related with part of I-309 and TNF-α production after LPS treatment of monocytes. Only SB203580 and SP600125 could reduce LPS-induced IP-10 expression in monocytes. Our data showed that inhibition of JNK activation by imidapril might at least in part involve in the suppressive effects of imidapril on the chemokines’ expression of LPS-stimulated monocytes. Data of Western blot and MAPK assay showed that imidapril was also able to downregulate LPS-induced p-MKK4 and p-c-Jun expression. These data suggested that ACEIs might reduce LPS-induced TNF-α, I-309, and IP-10 expression via, at least partly, the MKK4-JNK-c-Jun pathway.

Our use of imidapril and perindopril are prodrugs. Imidapril will be converted into imidaprilate, with bioavailability approximately 40%.^22–24 The time to peak concentration and half-life of imidapril are approximately 1 to 2 hours and 1 to 2.5 hours, respectively. Perindopril is the free acid form of perindopril erbumine, is a prodrug, and is metabolized in vivo by hydrolysis of the ester group to form perindoprilat. Imidapril, perindopril, and their active forms will be in our blood subsequently after ingestion. The plasma concentration of imidapril approximately 0 to 20 ng/mL (0-500 μM) after taking 2.5 to 20 mg of imidapril during 24 hours.^22–24 Therefore, the concentrations (0.0001-0.1 μM) in our experiment are relevant to the therapeutic concentrations achieved in patients.

Many different cytokines and chemokines may regulate Th2 immunity, and also, some cytokine and chemokine are produced by Th2 cells or are involved in Th2 polarization or effector functions. Therefore, it is difficult to evaluate all these cytokines and chemokines. Although imidapril gave a small, although significant reduction of TNF-α from monocytes (Fig. 1B) and IP-10 (Fig. 2B), it gave a significantly more impressive reduction of the Th2-recruiting chemokine, I-309 (Fig. 3B). Our data would suggest that imidapril may have a focused action on I-309 and therefore may influence Th2 responses because these cells express the receptor for I-309. The receptor for I-309 is CCR4. A number of other chemokines including TARC, MIP-1α, RANTES, and MCP-1 are ligands for this receptor. However, monocytes could not produce TARC well after stimulation (data not shown). We also did the same study about their effect on MIP-1α, MCP-1, and RANTES production. However, imidapril and perindopril had no significant effect on LPS-induced MIP-1α, MCP-1, and RANTES production (data not shown). Interleukins 4 and 10 are also important Th2-related cytokines. Interleukin 4 could not be produced well by human monocytes. Interleukin 10 is a late cytokine and is produced after TNF-α, probably as an autocrine method of regulating proinflammatory cytokine release by cells. Therefore, we also performed the same study about the effect on IL-10 production at 24- and 48-hour time points. However, imidapril and perindopril had no significant effect on LPS-IL-10 production at 24- and 48-hour time points (data not shown).

Angiotensin-converting enzyme inhibitors do not always have anti-inflammatory effects. Dandona et al.²⁵ report the first publication to demonstrate the anti-inflammatory and reactive oxygen species suppression of an angiotensin receptor blocker (valsartan). However, simvastatin and quinapril at a high dose produce no similar effect. In the present study, imidapril and perindopril seemed to be potent in suppressing the production of proinflammatory cytokine and Th1- and Th2-associated chemokines in monocytes. Tumor necrosis factor α is a proinflammatory cytokine, and anti-TNF-α therapies have clinical benefit for treating moderate to severe rheumatoid arthritis, Crohn disease, and other chronic inflammatory disorders.⁷ Th1- and also Th2-type immunoregulations play important roles in the pathogenesis of inflammation in autoimmune-related and allergic diseases. Therefore, except for the property of antihypertensive agents, ACEIs may have anti/proinflammatory, antiautoimmune, and antiallergic effects via suppressions on proinflammatory cytokine's and Th1- and Th2-related chemokines’ expression of monocytes. The ACEI may be a potent immunomodulatory agent and a good indication for hypertension related to inflammatory disorders.

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