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IL-6 plays an essential role in neutrophilia under inflammation
Cytokine 54 (2011) 92–99
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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
IL-6 plays an essential role in neutrophilia under inflammation Misato Hashizume a, Yoshinobu Higuchi b, Yasushi Uchiyama a, Masahiko Mihara a,⇑ a b
Product Research Department, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Japan Pharmaceutical Research Department 1, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Japan
a r t i c l e
i n f o
Article history: Received 22 June 2010 Received in revised form 5 January 2011 Accepted 7 January 2011 Available online 2 February 2011 Keywords: CD162 IL-6 Neutrophil Neutrophilia
a b s t r a c t In the present study, we explored the involvement of interleukin-6 (IL-6) in neutrophilia under inflammatory conditions. The neutrophil count in the peripheral blood was high in arthritic monkeys, and anti-IL-6 receptor antibody reduced neutrophil counts to normal levels. IL-6 injection into normal monkeys significantly increased neutrophil counts in the blood 3 h after injection. The expression of cluster of differentiation (CD) 162 on circulating neutrophils was reduced by IL-6 injection. IL-6 treatment in vitro did not affect CD162 expression on neutrophils from human blood. In IL-6-treated monkeys, IL-8 and granulocyte–macrophage colony-stimulating factor (GM-CSF) levels in plasma were clearly elevated. IL-8 and GM-CSF treatment in vitro reduced cell-surface CD162 expression on human neutrophils, and moreover, increased soluble CD162 expression in the cell supernatant. The addition of IL-6 into human whole peripheral blood induced IL-8 production and reduced CD162 expression on neutrophils. Furthermore, IL-8 and GM-CSF augmented mRNA expression of a disintegrin and metalloprotease like domain 10 (ADAM10) in neutrophils. Knock-down of ADAM10 by siRNA in neutrophil-like HL-60 cells partially reversed the expression of CD162 reduced by GM-CSF and IL-8 on HL-60 cells. In conclusion, IL-6 induced neutrophilia and reduced CD162 expression on neutrophils in inflammation. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction In rheumatoid arthritis (RA) patients, many neutrophils migrate into the inflamed joints and the number of neutrophils in the circulating blood is substantially increased. Neutrophils play an essential role in the course of inflammation, and removal of neutrophils by column or depletion of neutrophils ameliorates joint inflammation in RA patients and several animal arthritis models [1–3]. Migrated neutrophils are deeply involved in the onset and maintenance of inflammation through their robust production of inflammatory mediators such as prostaglandins, reactive oxygen species, complements, proteases and cytokines. The mechanisms underlying the trans-migration of neutrophils into inflamed sites have been actively investigated, and it is clear that chemokines and adhesion molecules are involved in neutrophil trans-migration [4]. Neutrophilia also facilitates the trans-migration of neutrophils into affected tissues; however, the precise mechanisms by which neutrophilia induces this trans-migration are not fully understood in RA patients. The mechanism of neutrophilia is thought to be the mobilization of neutrophils from the marginal pool to the circulation [5].
⇑ Corresponding author. Address: Product Research Department, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. Tel.: +81 550 87 6379; fax: +81 550 87 6782. E-mail address: [email protected] (M. Mihara). 1043-4666/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2011.01.007
In the marginal pool, leukocyte rolling and adhesion is mediated by the sequential interaction of adhesion molecules on the leukocyte surface with counter-ligands on the vascular endothelium and extra-vascular structures [6]. Constitutive expression of Pselectin and ICAM-1 on vascular endothelial cells is sufficient to support neutrophil rolling and adhesion, respectively [7,8]. Once neutrophils are activated, the expression levels of receptor molecules (CD162/PSGL-1, CD62L/L-selectin, and CD11b) can change depending on their exposure to stimulants. Following stimulation, neutrophils translocate from the marginal pool to the circulating blood. For example, G-CSF increases the neutrophil count in circulating blood by reducing CD62L expression, thus decreasing the rolling of neutrophils on the endothelium [3]. IL-6 is a pro-inflammatory cytokine and is produced by several kinds of cells in affected tissues. IL-6 participates in neutrophil migration by inducing production of chemokines such as IL-8 and MCP-1 and by inducing expression of adhesion molecules such as ICAM-1 on endothelial cells [9,10]. IL-6 blockade reduces infiltration of neutrophils into arthritic joints in a monkey model of arthritis [11]. Moreover, interestingly, IL-6 injection rapidly causes neutrophilia [12,13]. However, there are few reports indicating how IL-6 increases neutrophilia. Suwa et al. reported that IL-6 decreased CD62L expression on neutrophils and promoted their trafficking in rabbits [14]. However, in their study, they did not address the discrepancy between the effect of IL-6 on CD62L expression in vivo and that in vitro, nor did they verify the involve-
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ment of other adhesion molecules in neutrophilia induced by IL-6. In the present study, we studied the involvement of IL-6 in neutrophilia induction under inflammatory conditions.
the femoral vein with a syringe containing heparin sodium. Neutrophil count was measured with the Advia 120 hematology system.
2. Materials and methods
2.5. Plasma cytokine measurement
2.1. Reagents
The plasma concentrations of IL-8 and GM-CSF were measured using a Milliplex MAP Non-Human Primate Cytokine Panel – Premixed 23 Plex (Millipore, Billerica, MA) according to the manufacturer’s instructions. The data were analyzed with Bio-Plex Manager software v 5.0 (Bio-Rad, Hercules, CA).
Recombinant humanized anti-human IL-6 receptor (IL-6R) antibody (tocilizumab) and human IL-6 were prepared in our laboratories as previously described [15,16]. Cynomolgus monkey IL-6 cDNA was cloned from cynomolgus monkey thymus by PCR and transfected into Chinese hamster ovary cells, and the recombinant cynomolgus monkey IL-6 was purified by gel filtration. Recombinant human IL-8 and GM-CSF were purchased from Wako Pure Chemical Industries, Osaka, Japan. 2.2. Animals Cynomolgus monkeys (Macaca fascicularis, 3–5 years old) were purchased from Guangdong Scientific Instruments & Materials Import/Export Corporation (Guangzhou, China), Wing Freight Agent Co., Ltd. (Beijing, China), China National Scientific Instruments & Materials Import/Export Corporation (Beijing, China), Gaoyao Kangda Laboratory Animals Science & Technology Co., Ltd. (Guangdong, China), and Hamli Co., Ltd. (Ibaraki, Japan). Solid food was provided to each animal daily and water was available ad libitum from an automatic supply. All procedures involving animals were performed in accordance with standards published by the National Research Council (Guide for the Care and Use of Laboratory Animals) and by the National Institutes of Health (Public Health Service Policy on Human Care and Use of Laboratory Animals). The experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Chugai Pharmaceutical Co., Ltd. 2.3. Induction of arthritis Monkey collagen-induced arthritis (CIA) was induced by modifying the method that we reported previously [11]. Briefly, 4 mg/ mL bovine type II collagen (CII; Collagen Research Center, Tokyo, Japan) was emulsified in an equal volume of Freund’s complete adjuvant (Becton Dickinson, Grayson, GA). Under anesthesia with ketamine, five female cynomolgus monkeys (1.9–2.4 kg) were each injected intradermally with 2 mL of the emulsion at a dorsal site (1st immunization). Three weeks later, the animals were boosted with CII in the same manner (2nd immunization). At 2 weeks after the 2nd immunization (after onset of arthritis), a single intravenous injection of tocilizumab (30 mg/kg) was given. Blood was drawn at days 0, 11, 21, 28, 35, 36, 38, 42, 49, 56, and 63 from the femoral vein, with a syringe containing heparin sodium. Neutrophil count was measured with an Advia 120 hematology system (Siemens Healthcare Diagnostics, Deerfield, IL). Arthritis (swelling of joints) was assessed by measuring the longitudinal and transverse axes of the proximal interphalangeal joints (PIP joints) of the fore and hind limbs (excluding the thumb) with vernier calipers and calculating the oval area of each PIP joint. 2.4. Treatment of cynomolgus monkeys with IL-6 Three male cynomolgus monkeys (3.2–3.6 kg) were treated with a single subcutaneous injection of cynomolgus monkey IL-6 (5 lg/kg) or the same volume of phosphate buffered saline (PBS) Tocilizumab (8 mg/kg) was injected intravenously 24 h before the IL-6 injection. Blood was drawn at 3 h after IL-6 injection from
2.6. Flow cytometric analysis of monkey neutrophils Cynomolgus monkey whole blood (100 lL) was incubated with 20 lL each of anti-CD62L antibody (clone MA1-81194; Thermo Fisher Scientific, Waltham, MA), anti-CD11b antibody (clone ICRF44; BD Bioscience, Franklin Lakes, NJ), anti-CD162 antibody (clone KPL-1; BD Bioscience), anti-IL-6R antibody (clone M5; BD Bioscience), or anti-CD66b antibody (granulocyte marker, clone G10F5; BD Bioscience) for 30 min at room temperature. The red blood cells were then lysed and suspended in BD FACS sheath solution (BD Bioscience). Expressions of CD62L, CD11b, CD162, and IL-6R on CD66b positive cells (referred to here as neutrophils) were detected by flow cytometer (FACSCanto II; BD Bioscience), and the calculated mean fluorescence intensities (MFI) were analyzed using FACSDiva software (BD Bioscience). 2.7. In vitro study using purified human neutrophils Human venous blood was obtained from three healthy volunteers who had given their informed consent. Human venous blood collected in heparin sodium was centrifuged using a Polymorphprep (Axis-Shield PoC AS, Oslo, Norway), and contaminating red blood cells were eliminated by hypotonic lysis. After washing, the neutrophils were suspended in RPMI-1640 medium (Gibco, Glandisland, NY) containing 10% fetal bovine serum Neutrophils (purity > 95%; 1 105 cells/0.5 mL-well) were then incubated in 24-well plates with IL-6 (10 ng/mL), IL-8 (10 ng/mL), and GM-CSF (10 ng/mL) for 2 h. After incubation, cells and supernatants were collected, and expressions of CD162, CD11b, CD62L, and IL-6R on the CD66b positive cells were detected by FACSCanto II, and the concentration of soluble CD162 (sCD162) was measured by ELISA (Promokine, Heidelberg, Germany). 2.8. In vitro study using human whole blood Human whole blood from the three healthy subjects (500 lL) was incubated in a 24-well plate with IL-6 (10 ng/mL), tocilizumab (100 lg/mL), and anti-IL-8 antibody (Sigma Aldrich, St. Louis, MO; 500 lg/mL) for 2 h. After incubation, cells and plasma were collected, and expression of CD162 was detected by FACSCanto II and the concentration of IL-8 and GM-CSF was measured by ELISA (Thermo Fisher Scientific, Waltham, MA and R&D systems, Minneapolis, MN, respectively). 2.9. Quantitative real-time PCR Human neutrophils were incubated in 24-well plates with IL-6, IL-8 and GM-CSFfor 2 h. After incubation, total RNA was extracted using an RNeasy kit (Qiagen, Valencia, CA). Synthesis of cDNA was performed using an Omniscript RT kit (Qiagen) with nine random primers (TaKaRa, Shiga, Japan) according to the manufacturer’s protocol. Quantitative real-time PCR was performed by running a TaqMan gene expression assay (Applied Biosystems, Foster City,
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CA), targeting human ADAM8, ADAM10, ADAM17, and human glyceraldehyde-3-phosphate dehydrogenase, on an ABI PRISM 7500 system (Applied Biosystems) according to the manufacturer’s protocol. Thermal cycle parameters were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of amplification with 15 s at 95 °C for denaturation, 1 min at 60 °C for annealing and elongation. 2.10. Culture of HL-60 cells and ADAM10 siRNA transfection HL-60 cells (ATCC, Manassas, VA; 5 105 cells/mL) were cultured in RPMI-1640 supplemented with 10% FBS and were subcultured at 2–3 day intervals. To differentiate HL-60 cells toward a neutrophil-like phenotype, HL-60 cells were treated with DMSO as reported previously [17]. Briefly, an aliquot of 5 105 cells/mL was cultured in 10 mL of RPMI-1640 medium and incubated in the presence of 1.25% (v/v) DMSO for 4 days in a T75 flask. Every 2 days, the medium was changed and the number of cells adjusted to 5 105 cells/mL. After 4 days’ culture, cell morphology changed to giant cells of irregular shape. After 4 days’ culture with DMSO, the HL-60 cells were transfected with control siRNA and ADAM10 siRNA by utilizing the Accell siRNA delivery protocol (Thermo Fisher Scientific). The HL-60 cells were aliquoted into a 96-well plate (3 105 cells/well) in Accell delivery medium containing 1 lM siRNA. Cells were incubated with IL-8 (10 ng/mL) and GM-CSF (10 ng/mL) for 3 days, and were collected for western blotting and flow cytometric analysis. 2.11. Western blotting for ADAM10 Cells were lysed with lysis buffer (25 mM Tris–HCl pH 7.4, 25 mM NaF, 1 mM Na3VO4, 1 mM DTT, 1 tablet of protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) 1% Triton X100). Cell lysates were separated by 15% sodium dodecyl sulfate– polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). After blocking with 3% BSA in TBS for 1 h at room temperature, rabbit polyclonal antibody to human ADAM10 (Sigma Aldrich) or mouse monoclonal antibody to b-actin (Sigma Aldrich) in 1% BSA/TBS (1:2000 dilution for each) was added and incubated for 16–20 h at 4 °C. After 1 h incubation with the secondary antibody, binding was visualized by using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) with a ChemiDoc XRS digital imaging system (Bio-Rad Laboratories, Hercules, CA) and was analyzed by using Quantity One software (Bio-Rad Laboratories). 2.12. Statistical analysis Statistical significances were calculated by unpaired t-test using the Statistical Analysis System software package (SAS Institute Japan, Tokyo, Japan). The significance level was set to 5%. 3. Results 3.1. Anti-IL-6 receptor antibody decreased neutrophil counts in arthritic monkeys The neutrophil counts increased rapidly in the first week after the 1st immunization, and then remained in a steady state until week 5. The elevated neutrophil counts were rapidly decreased by tocilizumab treatment (Fig. 1). After the initial rapid decrease, neutrophil counts gradually began to increase. In this model, arthritis was first observed at around day 28 and swelling of joints reached the maximum at day 35. We previously reported that serum IL-6 levels were clearly elevated after the 1st
Fig. 1. Effect of tocilizumab on blood neutrophil count in arthritic monkeys. Monkeys were immunized twice with bovine type II collagen with a 3-week interval between injections. Five weeks after the 1st collagen immunization, tocilizumab (30 mg/kg) was injected intravenously. Neutrophil counts were measured by the Advia 120 hematology system. Each point represents the mean (n = 8) and SE. Arrow indicates tocilizumab injection.
immunization and that tocilizumab treatment reduced swelling of joints [11]. In the present study, similar results were reproduced (data not shown). 3.2. IL-6 injection increased neutrophil counts and decreased CD162 expression in monkeys We examined whether IL-6 increased neutrophil counts in monkeys and whether IL-6 affected the expression of adhesion molecules on neutrophils. Neutrophil counts in normal monkeys were 2–5 103 cells/mm3. Neutrophil counts in blood were significantly increased 3 h after IL-6 injection (14.2 ± 3.8 cells/mm3; Fig. 2A). By 8 h after IL-6 injection, the neutrophil counts had dropped to 8.8 ± 2.4 cells/mm3. Pre-treatment with tocilizumab (24 h before IL-6 injection) completely inhibited the IL-6-induced increase in the neutrophil counts at 3 h after IL-6 injection. Clear expression of CD62L, CD11b, CD162, and IL-6R in neutrophils was observed in PBS-treated monkeys (Fig. 2B). CD162 expression was markedly reduced on neutrophils from IL-6-treated monkeys, but expressions of CD62L, CD11b, and IL-6R were not influenced (Fig. 2C). 3.3. IL-6 did not directly affect adhesion molecule expression on isolated human neutrophils We examined whether IL-6 directly reduced CD162 expression. Isolated human neutrophils were incubated with IL-6 for 2 h, and then CD11b, CD62L, and CD162 expression levels were analyzed. Human neutrophils also expressed IL-6R, and IL-6 induced cytokine production from human neutrophils (data not shown). Unexpectedly, IL-6 did not affect the expression of CD62L, CD11b, or CD162 on neutrophils at all (Fig. 3). 3.4. Involvement of GM-CSF and IL-8 in reduction of CD162 expression by IL-6 From the above results, we hypothesized that IL-6 indirectly reduced adhesion molecule expression via cytokine or chemokine production. To examine this idea, we measured plasma cytokine levels in IL-6-treated monkeys. Plasma concentration of IL-6 was increased by IL-6 injection and it was similar to that in CIA mon-
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Fig. 2. IL-6 increased neutrophil counts in blood and reduced adhesion molecule expression on neutrophils in monkeys. Monkeys were subcutaneously injected with cynomolgus monkey IL-6 (5 lg/kg) or PBS. Tocilizumab (8 mg/kg) was injected intravenously 24 h before IL-6 injection. At 3 h after IL-6 injection, peripheral blood was drawn. (A) Neutrophil counts in peripheral blood. (B) Representative expression levels of CD62L, CD11b, CD162, and IL-6R on neutrophils. Shaded histograms indicate the control IgG (isotype control); dotted lines indicate expression levels in a PBS-treated monkey; solid lines indicate expression levels in an IL-6-treated monkey. (C) Mean fluorescence intensities for CD11b, CD62L, CD162, and IL-6R. Each column and error bar indicates the mean and SE of three animals. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. PBS).
Fig. 3. IL-6 did not affect expression of adhesion molecules on human neutrophils. Human neutrophils were isolated from the peripheral blood of healthy subjects. Cells were incubated with IL-6 (10 ng/mL) for 2 h. After culture, expression of CD11b, CD62L, and CD162 on neutrophils was analyzed by the flow cytometry system. Data show the mean fluorescence intensities. Each column and error bar indicates the mean and SE of three donors.
keys as previously reported ( 300 pg/mL) [11]. Plasma concentrations of IL-8 and GM-CSF were increased by IL-6 injection, and tocilizumab inhibited IL-6-induced IL-8 and GM-CSF production (Fig. 4A). G-CSF expression was not induced by IL-6 injection (data not shown). On the other hand, tocilizumab increased blood IL-6 levels. Next, we examined the effect of IL-8 and GM-CSF on CD162 expression. As expected, IL-8 and GM-CSF significantly decreased CD162 expression on neutrophils (Fig. 4B). The combination of IL-8 and GM-CSF did not show a clear additive effect on the reduction of CD162 expression. The concentrations of the soluble form of CD162 were significantly higher in supernatants of neutrophils stimulated with GMCSF and IL-8 than in the supernatant from the non-treated group (Fig. 4C). We further studied whether a similar phenomenon would be observed in the case of human whole blood. Human whole blood was incubated with IL-6 for 2 h, followed by measurement of IL8 and GM-CSF in the cell supernatants. IL-8, but not GM-CSF, was induced by IL-6 (Fig. 5A). IL-6 also decreased CD162 expression
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Fig. 4. GM-CSF and IL-8 reduced CD162 expression on human neutrophils. (A) Serum levels of IL-8, GM-CSF and IL-6 in IL-6-treated monkeys were measured by using a Milliplex MAP Non-Human Primate Cytokine Panel. Each column and error bar indicates the mean and SE of three animals. Statistical significance was analyzed by unpaired ttest (p < 0.05 vs. PBS treated). (B) Human neutrophils were incubated for 2 h in the presence of IL-6 (10 ng/mL), GM-CSF (10 ng/mL), and IL-8 (10 ng/mL). After incubation, CD162 expression was measured by the flow cytometry system. Each column and error bar indicates the mean and SE of three donors. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. medium control). (C) Soluble CD162 levels in cell supernatants. Each column and error bar indicates the mean and SE of three donors. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. medium control).
Fig. 5. Human whole blood study. Human whole blood from three healthy subjects (500 lL) was incubated in a 24-well plate with IL-6 (10 ng/mL), tocilizumab (100 lg/mL), and anti-IL-8 antibody (500 lg/mL) for 2 h. After incubation, cells and plasma were collected. (A) The concentration of IL-8 in IL-6-treated whole blood. (B) Expression of CD162 on neutrophils. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. medium control).
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on neutrophils in whole blood, and the reduction of CD162 expression by IL-6 was reversed by tocilizumab and anti-IL-8 antibody, which restored CD162 expression to control levels (Fig. 5B). 3.5. GM-CSF and IL-8 induced expression of the cleavage enzyme ADAM10 that mediates CD162 shedding in isolated human neutrophils The fact that sCD162 increased in the supernatants of neutrophils stimulated with GM-CSF and IL-8 (Fig. 4C) suggested the cleavage of membrane-bound CD162 by a certain enzyme. ADAM8, ADAM10, and ADAM17 have all been shown to contribute to the shedding of adhesion molecules [18,19]. The role of ADAM proteins in CD162 shedding is not well characterized for any type of cell. To determine which ADAM family is involved in CD162 shedding, we examined ADAM8, ADAM10, and ADAM17 mRNA expression in neutrophils after stimulation by IL-6, IL-8, and GM-CSF. IL-8 and GM-CSF induced transcription of ADAM10, but not of ADAM8 or ADAM17, and IL-6 did not affect any of the ADAM8, ADAM10, or ADAM17 mRNA levels (Fig. 6A). 3.6. ADAM10 is involved in CD162 shedding in HL-60 cells To clarify the involvement of ADAM10 in CD162 shedding, we silenced ADAM10 mRNA with small interfering RNA (siRNA). In this study we used cells of the neutrophil-like cell line HL-60 because it takes several days to transfect siRNA into cells and isolated human neutrophils rapidly undergo apoptosis. HL-60 cells can be differentiated to a neutrophil-like phenotype by treatment with DMSO [17]. CD162 expression was induced in HL-60 cells treated with DMSO (HL-60D cells), compared with CD162 expression in HL-60 cells (Fig. 6B). GM-CSF and IL-8 induced ADAM10 protein in HL-60D cells, and ADAM10 siRNA inhibited induction of ADAM10 by GM-CSF and IL-8 (Fig. 6C). We further examined whether GM-CSF and IL-8 induced CD162 shedding. In control HL-60D cells, GM-CSF and IL-8 induced CD162 shedding like in isolated human neutrophils. On the other hand, in HL-60D cells with ADAM10 knocked-down, CD162 shedding was partially inhibited by GM-CSF and IL-8 (Fig. 6D). 4. Discussion The present report describes one mechanism of neutrophilia in arthritis. We showed that (1) IL-6 blockade rapidly improved neutrophilia in arthritic monkeys; (2) IL-6 injection increased neutrophil counts and remarkably reduced CD162 expression on circulating neutrophils in monkeys; (3) IL-6 injection induced GM-CSF and IL-8 production in monkeys; (4) GM-CSF and IL-8 reduced CD162 expression in human isolated neutrophils, but IL-6 itself did not; and (5) GM-CSF and IL-8 promoted shedding of CD162 in neutrophils via ADAM10 induction. These findings suggest that IL-6 is greatly involved in neutrophilia in arthritis. Moreover, IL-6 indirectly reduced expression of CD162 in neutrophils by inducing ADAM10 production from neutrophils. In this study, we showed that neutrophil counts were increased by collagen immunization and that high neutrophil counts were maintained after the onset of arthritis, and we showed that tocilizumab injection rapidly reduced the elevated neutrophil count to normal level. Moreover, elevated production of IL-6 is observed in the monkey CIA model [11]. These lines of evidence suggest that in this arthritis model, circulating neutrophils are increased by IL6. Tocilizumab reduces joint swelling and migration of neutrophils into inflamed joints [11], suggesting that a reduction in the number of blood neutrophils leads to improvement in joint swelling. Blood neutrophil counts are controlled by the differentiation and proliferation of precursor cells and by trafficking from the
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bone marrow and the marginal pool. It takes 7–10 days for myeloblastic and myelocytic progenitors to differentiate into neutrophils, but on the other hand, trafficking of neutrophils occurs rapidly [5]. IL-6 contributes to differentiation from progenitors to neutrophils [20]. Indeed, we previously reported that Gr-1 positive cells (neutrophils) in bone marrow and peripheral blood neutrophils significantly increase in IL-6 transgenic mice [21]. However, in this study IL-6 increased neutrophil counts very rapidly. These facts strongly suggest that IL-6 promoted the movement of neutrophils from the marginal pool to the blood. To the best of our knowledge, this is the first report demonstrating both in vivo and in vitro that IL-6 reduces CD162 expression on neutrophils. CD62L and CD162 are constitutively expressed on most circulating leukocytes, and they have been shown to play a role in leukocyte–endothelial interactions [22]. It is reported that IL-6 reduces CD62L expression on circulating neutrophils in rabbits [14]. However, as we also found in this study, it has been reported that changes in CD162 expression are more strongly correlated with neutrophil increase than changes in CD62L expression [23]. Isolated neutrophils expressed membrane-bound IL-6R, and IL6 induced IL-23 production from neutrophils (data not shown), demonstrating that neutrophils could respond to IL-6. However, IL-6 did not directly reduce CD162 expression on neutrophils. These results led us to hypothesize that cytokines or chemokines induced by IL-6 changed the expression of CD162. As expected, IL-6 injection induced IL-8 and GM-CSF production in monkeys, and these cytokines were found to be involved in reduction of CD162 expression on isolated neutrophils and in whole blood. However, it is necessary to perform further studies in order to conclude that IL-8 and GM-CSF reduces CD162 expression in vivo. It is reported that G-CSF also increases neutrophil counts and reduces CD162 expression on neutrophils in humans [23]. However, IL-6 did not increase serum G-CSF concentrations in monkeys. Therefore, the mechanism for the IL-6-induced decrease of CD162 expression might have no involvement with G-CSF. IL-6 injection induced GM-CSF and IL-8 production in monkeys. On the other hand, IL-6 induced IL-8 but not GM-CSF production from human peripheral whole blood in vitro. IL-8 and GM-CSF are produced by many cell types, including activated T and B cells, macrophages, monocytes, endothelial cells, and fibroblasts. We previously reported that IL-6 induces IL-8 in mononuclear cells of peripheral blood [9]. The difference between the in vivo and in vitro results may be explained as follows: IL-8 was induced by IL-6 from cells existing in circulating blood, but GM-CSF was from non-hematopoietic cells. In RA patients, serum concentrations of IL-6, IL-8, and GM-CSF are higher than those of healthy subjects [24]. Therefore, it is likely that IL-6 induces IL-8 and GM-CSF production in RA patients. GM-CSF and IL-8 decreased the membrane-bound CD162 and increased production of soluble CD162. This result strongly suggested that membrane-bound CD162 was cleaved. ADAM10 is expressed in most cells and plays the role of a secretase for numerous membrane proteins, including CD44, TNF, and E-cadherin [25]. It has been reported that ADAM10 is involved in CD162 shedding in monocytes [26]. In this study, we identified ADAM10 as a CD162 sheddase in neutrophils. However, because the influence of ADAM10 siRNA was partial, it is possible that another sheddase also mediates CD162 shedding from neutrophils. However, it is still unclear whether CD162 is essential for IL-6-induced neutrophilia. Further study should be performed to resolve this issue. Anti-IL-6R antibody (tocilizumab) treatment dramatically improves the symptoms of RA and normalizes neutrophil counts [27]. Our present study clearly suggests that over-production of IL-6 increases neutrophil counts in blood via the down-regulation of CD162 in RA patients, and that IL-6 blockade inhibits increased
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Fig. 6. ADAM10 mediated CD162 shedding from HL-60 cells. (A) Human neutrophils were incubated for 2 h in the presence of IL-6 (10 ng/mL), GM-CSF (10 ng/mL), and IL-8 (10 ng/mL). After incubation, mRNA levels of ADAM8, ADAM10, and ADAM17 were measured by real-time PCR. The ADAM expression by non-treated controls was defined as 1. Each column and error bar indicates the mean and SD of three donors. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. medium control). HL-60 cells were cultured with DMSO for 4 days and then cells were transfected with control siRNA and ADAM10 siRNA following the protocol of the transfection kit’s manufacturer. siRNA-treated cells were incubated with IL-8 (10 ng/mL) and GM-CSF (10 ng/mL) for 4 days. (B) CD162 expression in DMSO-treated HL-60 cells (HL-60D). Shaded histograms indicate non-treated HL-60 cells; solid lines indicate HL-60D cells. (C) ADAM10 expression of siRNA-treated HL-60D cells. ADAM10 and b-actin (internal control) protein expressions were measured by western blotting. Each column and error bar indicates the mean (n = 3) and SD. Statistical significance was analyzed by unpaired t-test (, #p < 0.05). (D) Effect of GM-CSF and IL-8 on CD162 in HL-60D cells. Shaded histograms indicate non-stimulated/control siRNA cells; solid lines indicate non-stimulated/ ADAM10 siRNA cells; thick lines indicate stimulated/control siRNA cells; dotted lines indicate stimulated/ADAM10 siRNA cells.
neutrophil counts induced by IL-6. Further studies in RA patients are warranted to confirm the results we obtained from the animal model.
In conclusion, IL-6 induces neutrophilia by promoting the production of IL-8 and GM-CSF which reduce expression of adhesion molecules on neutrophils.
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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
IL-6 plays an essential role in neutrophilia under inflammation Misato Hashizume a, Yoshinobu Higuchi b, Yasushi Uchiyama a, Masahiko Mihara a,⇑ a b
Product Research Department, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Japan Pharmaceutical Research Department 1, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Japan
a r t i c l e
i n f o
Article history: Received 22 June 2010 Received in revised form 5 January 2011 Accepted 7 January 2011 Available online 2 February 2011 Keywords: CD162 IL-6 Neutrophil Neutrophilia
a b s t r a c t In the present study, we explored the involvement of interleukin-6 (IL-6) in neutrophilia under inflammatory conditions. The neutrophil count in the peripheral blood was high in arthritic monkeys, and anti-IL-6 receptor antibody reduced neutrophil counts to normal levels. IL-6 injection into normal monkeys significantly increased neutrophil counts in the blood 3 h after injection. The expression of cluster of differentiation (CD) 162 on circulating neutrophils was reduced by IL-6 injection. IL-6 treatment in vitro did not affect CD162 expression on neutrophils from human blood. In IL-6-treated monkeys, IL-8 and granulocyte–macrophage colony-stimulating factor (GM-CSF) levels in plasma were clearly elevated. IL-8 and GM-CSF treatment in vitro reduced cell-surface CD162 expression on human neutrophils, and moreover, increased soluble CD162 expression in the cell supernatant. The addition of IL-6 into human whole peripheral blood induced IL-8 production and reduced CD162 expression on neutrophils. Furthermore, IL-8 and GM-CSF augmented mRNA expression of a disintegrin and metalloprotease like domain 10 (ADAM10) in neutrophils. Knock-down of ADAM10 by siRNA in neutrophil-like HL-60 cells partially reversed the expression of CD162 reduced by GM-CSF and IL-8 on HL-60 cells. In conclusion, IL-6 induced neutrophilia and reduced CD162 expression on neutrophils in inflammation. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction In rheumatoid arthritis (RA) patients, many neutrophils migrate into the inflamed joints and the number of neutrophils in the circulating blood is substantially increased. Neutrophils play an essential role in the course of inflammation, and removal of neutrophils by column or depletion of neutrophils ameliorates joint inflammation in RA patients and several animal arthritis models [1–3]. Migrated neutrophils are deeply involved in the onset and maintenance of inflammation through their robust production of inflammatory mediators such as prostaglandins, reactive oxygen species, complements, proteases and cytokines. The mechanisms underlying the trans-migration of neutrophils into inflamed sites have been actively investigated, and it is clear that chemokines and adhesion molecules are involved in neutrophil trans-migration [4]. Neutrophilia also facilitates the trans-migration of neutrophils into affected tissues; however, the precise mechanisms by which neutrophilia induces this trans-migration are not fully understood in RA patients. The mechanism of neutrophilia is thought to be the mobilization of neutrophils from the marginal pool to the circulation [5].
⇑ Corresponding author. Address: Product Research Department, Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. Tel.: +81 550 87 6379; fax: +81 550 87 6782. E-mail address: [email protected] (M. Mihara). 1043-4666/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2011.01.007
In the marginal pool, leukocyte rolling and adhesion is mediated by the sequential interaction of adhesion molecules on the leukocyte surface with counter-ligands on the vascular endothelium and extra-vascular structures [6]. Constitutive expression of Pselectin and ICAM-1 on vascular endothelial cells is sufficient to support neutrophil rolling and adhesion, respectively [7,8]. Once neutrophils are activated, the expression levels of receptor molecules (CD162/PSGL-1, CD62L/L-selectin, and CD11b) can change depending on their exposure to stimulants. Following stimulation, neutrophils translocate from the marginal pool to the circulating blood. For example, G-CSF increases the neutrophil count in circulating blood by reducing CD62L expression, thus decreasing the rolling of neutrophils on the endothelium [3]. IL-6 is a pro-inflammatory cytokine and is produced by several kinds of cells in affected tissues. IL-6 participates in neutrophil migration by inducing production of chemokines such as IL-8 and MCP-1 and by inducing expression of adhesion molecules such as ICAM-1 on endothelial cells [9,10]. IL-6 blockade reduces infiltration of neutrophils into arthritic joints in a monkey model of arthritis [11]. Moreover, interestingly, IL-6 injection rapidly causes neutrophilia [12,13]. However, there are few reports indicating how IL-6 increases neutrophilia. Suwa et al. reported that IL-6 decreased CD62L expression on neutrophils and promoted their trafficking in rabbits [14]. However, in their study, they did not address the discrepancy between the effect of IL-6 on CD62L expression in vivo and that in vitro, nor did they verify the involve-
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ment of other adhesion molecules in neutrophilia induced by IL-6. In the present study, we studied the involvement of IL-6 in neutrophilia induction under inflammatory conditions.
the femoral vein with a syringe containing heparin sodium. Neutrophil count was measured with the Advia 120 hematology system.
2. Materials and methods
2.5. Plasma cytokine measurement
2.1. Reagents
The plasma concentrations of IL-8 and GM-CSF were measured using a Milliplex MAP Non-Human Primate Cytokine Panel – Premixed 23 Plex (Millipore, Billerica, MA) according to the manufacturer’s instructions. The data were analyzed with Bio-Plex Manager software v 5.0 (Bio-Rad, Hercules, CA).
Recombinant humanized anti-human IL-6 receptor (IL-6R) antibody (tocilizumab) and human IL-6 were prepared in our laboratories as previously described [15,16]. Cynomolgus monkey IL-6 cDNA was cloned from cynomolgus monkey thymus by PCR and transfected into Chinese hamster ovary cells, and the recombinant cynomolgus monkey IL-6 was purified by gel filtration. Recombinant human IL-8 and GM-CSF were purchased from Wako Pure Chemical Industries, Osaka, Japan. 2.2. Animals Cynomolgus monkeys (Macaca fascicularis, 3–5 years old) were purchased from Guangdong Scientific Instruments & Materials Import/Export Corporation (Guangzhou, China), Wing Freight Agent Co., Ltd. (Beijing, China), China National Scientific Instruments & Materials Import/Export Corporation (Beijing, China), Gaoyao Kangda Laboratory Animals Science & Technology Co., Ltd. (Guangdong, China), and Hamli Co., Ltd. (Ibaraki, Japan). Solid food was provided to each animal daily and water was available ad libitum from an automatic supply. All procedures involving animals were performed in accordance with standards published by the National Research Council (Guide for the Care and Use of Laboratory Animals) and by the National Institutes of Health (Public Health Service Policy on Human Care and Use of Laboratory Animals). The experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Chugai Pharmaceutical Co., Ltd. 2.3. Induction of arthritis Monkey collagen-induced arthritis (CIA) was induced by modifying the method that we reported previously [11]. Briefly, 4 mg/ mL bovine type II collagen (CII; Collagen Research Center, Tokyo, Japan) was emulsified in an equal volume of Freund’s complete adjuvant (Becton Dickinson, Grayson, GA). Under anesthesia with ketamine, five female cynomolgus monkeys (1.9–2.4 kg) were each injected intradermally with 2 mL of the emulsion at a dorsal site (1st immunization). Three weeks later, the animals were boosted with CII in the same manner (2nd immunization). At 2 weeks after the 2nd immunization (after onset of arthritis), a single intravenous injection of tocilizumab (30 mg/kg) was given. Blood was drawn at days 0, 11, 21, 28, 35, 36, 38, 42, 49, 56, and 63 from the femoral vein, with a syringe containing heparin sodium. Neutrophil count was measured with an Advia 120 hematology system (Siemens Healthcare Diagnostics, Deerfield, IL). Arthritis (swelling of joints) was assessed by measuring the longitudinal and transverse axes of the proximal interphalangeal joints (PIP joints) of the fore and hind limbs (excluding the thumb) with vernier calipers and calculating the oval area of each PIP joint. 2.4. Treatment of cynomolgus monkeys with IL-6 Three male cynomolgus monkeys (3.2–3.6 kg) were treated with a single subcutaneous injection of cynomolgus monkey IL-6 (5 lg/kg) or the same volume of phosphate buffered saline (PBS) Tocilizumab (8 mg/kg) was injected intravenously 24 h before the IL-6 injection. Blood was drawn at 3 h after IL-6 injection from
2.6. Flow cytometric analysis of monkey neutrophils Cynomolgus monkey whole blood (100 lL) was incubated with 20 lL each of anti-CD62L antibody (clone MA1-81194; Thermo Fisher Scientific, Waltham, MA), anti-CD11b antibody (clone ICRF44; BD Bioscience, Franklin Lakes, NJ), anti-CD162 antibody (clone KPL-1; BD Bioscience), anti-IL-6R antibody (clone M5; BD Bioscience), or anti-CD66b antibody (granulocyte marker, clone G10F5; BD Bioscience) for 30 min at room temperature. The red blood cells were then lysed and suspended in BD FACS sheath solution (BD Bioscience). Expressions of CD62L, CD11b, CD162, and IL-6R on CD66b positive cells (referred to here as neutrophils) were detected by flow cytometer (FACSCanto II; BD Bioscience), and the calculated mean fluorescence intensities (MFI) were analyzed using FACSDiva software (BD Bioscience). 2.7. In vitro study using purified human neutrophils Human venous blood was obtained from three healthy volunteers who had given their informed consent. Human venous blood collected in heparin sodium was centrifuged using a Polymorphprep (Axis-Shield PoC AS, Oslo, Norway), and contaminating red blood cells were eliminated by hypotonic lysis. After washing, the neutrophils were suspended in RPMI-1640 medium (Gibco, Glandisland, NY) containing 10% fetal bovine serum Neutrophils (purity > 95%; 1 105 cells/0.5 mL-well) were then incubated in 24-well plates with IL-6 (10 ng/mL), IL-8 (10 ng/mL), and GM-CSF (10 ng/mL) for 2 h. After incubation, cells and supernatants were collected, and expressions of CD162, CD11b, CD62L, and IL-6R on the CD66b positive cells were detected by FACSCanto II, and the concentration of soluble CD162 (sCD162) was measured by ELISA (Promokine, Heidelberg, Germany). 2.8. In vitro study using human whole blood Human whole blood from the three healthy subjects (500 lL) was incubated in a 24-well plate with IL-6 (10 ng/mL), tocilizumab (100 lg/mL), and anti-IL-8 antibody (Sigma Aldrich, St. Louis, MO; 500 lg/mL) for 2 h. After incubation, cells and plasma were collected, and expression of CD162 was detected by FACSCanto II and the concentration of IL-8 and GM-CSF was measured by ELISA (Thermo Fisher Scientific, Waltham, MA and R&D systems, Minneapolis, MN, respectively). 2.9. Quantitative real-time PCR Human neutrophils were incubated in 24-well plates with IL-6, IL-8 and GM-CSFfor 2 h. After incubation, total RNA was extracted using an RNeasy kit (Qiagen, Valencia, CA). Synthesis of cDNA was performed using an Omniscript RT kit (Qiagen) with nine random primers (TaKaRa, Shiga, Japan) according to the manufacturer’s protocol. Quantitative real-time PCR was performed by running a TaqMan gene expression assay (Applied Biosystems, Foster City,
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CA), targeting human ADAM8, ADAM10, ADAM17, and human glyceraldehyde-3-phosphate dehydrogenase, on an ABI PRISM 7500 system (Applied Biosystems) according to the manufacturer’s protocol. Thermal cycle parameters were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of amplification with 15 s at 95 °C for denaturation, 1 min at 60 °C for annealing and elongation. 2.10. Culture of HL-60 cells and ADAM10 siRNA transfection HL-60 cells (ATCC, Manassas, VA; 5 105 cells/mL) were cultured in RPMI-1640 supplemented with 10% FBS and were subcultured at 2–3 day intervals. To differentiate HL-60 cells toward a neutrophil-like phenotype, HL-60 cells were treated with DMSO as reported previously [17]. Briefly, an aliquot of 5 105 cells/mL was cultured in 10 mL of RPMI-1640 medium and incubated in the presence of 1.25% (v/v) DMSO for 4 days in a T75 flask. Every 2 days, the medium was changed and the number of cells adjusted to 5 105 cells/mL. After 4 days’ culture, cell morphology changed to giant cells of irregular shape. After 4 days’ culture with DMSO, the HL-60 cells were transfected with control siRNA and ADAM10 siRNA by utilizing the Accell siRNA delivery protocol (Thermo Fisher Scientific). The HL-60 cells were aliquoted into a 96-well plate (3 105 cells/well) in Accell delivery medium containing 1 lM siRNA. Cells were incubated with IL-8 (10 ng/mL) and GM-CSF (10 ng/mL) for 3 days, and were collected for western blotting and flow cytometric analysis. 2.11. Western blotting for ADAM10 Cells were lysed with lysis buffer (25 mM Tris–HCl pH 7.4, 25 mM NaF, 1 mM Na3VO4, 1 mM DTT, 1 tablet of protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) 1% Triton X100). Cell lysates were separated by 15% sodium dodecyl sulfate– polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). After blocking with 3% BSA in TBS for 1 h at room temperature, rabbit polyclonal antibody to human ADAM10 (Sigma Aldrich) or mouse monoclonal antibody to b-actin (Sigma Aldrich) in 1% BSA/TBS (1:2000 dilution for each) was added and incubated for 16–20 h at 4 °C. After 1 h incubation with the secondary antibody, binding was visualized by using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) with a ChemiDoc XRS digital imaging system (Bio-Rad Laboratories, Hercules, CA) and was analyzed by using Quantity One software (Bio-Rad Laboratories). 2.12. Statistical analysis Statistical significances were calculated by unpaired t-test using the Statistical Analysis System software package (SAS Institute Japan, Tokyo, Japan). The significance level was set to 5%. 3. Results 3.1. Anti-IL-6 receptor antibody decreased neutrophil counts in arthritic monkeys The neutrophil counts increased rapidly in the first week after the 1st immunization, and then remained in a steady state until week 5. The elevated neutrophil counts were rapidly decreased by tocilizumab treatment (Fig. 1). After the initial rapid decrease, neutrophil counts gradually began to increase. In this model, arthritis was first observed at around day 28 and swelling of joints reached the maximum at day 35. We previously reported that serum IL-6 levels were clearly elevated after the 1st
Fig. 1. Effect of tocilizumab on blood neutrophil count in arthritic monkeys. Monkeys were immunized twice with bovine type II collagen with a 3-week interval between injections. Five weeks after the 1st collagen immunization, tocilizumab (30 mg/kg) was injected intravenously. Neutrophil counts were measured by the Advia 120 hematology system. Each point represents the mean (n = 8) and SE. Arrow indicates tocilizumab injection.
immunization and that tocilizumab treatment reduced swelling of joints [11]. In the present study, similar results were reproduced (data not shown). 3.2. IL-6 injection increased neutrophil counts and decreased CD162 expression in monkeys We examined whether IL-6 increased neutrophil counts in monkeys and whether IL-6 affected the expression of adhesion molecules on neutrophils. Neutrophil counts in normal monkeys were 2–5 103 cells/mm3. Neutrophil counts in blood were significantly increased 3 h after IL-6 injection (14.2 ± 3.8 cells/mm3; Fig. 2A). By 8 h after IL-6 injection, the neutrophil counts had dropped to 8.8 ± 2.4 cells/mm3. Pre-treatment with tocilizumab (24 h before IL-6 injection) completely inhibited the IL-6-induced increase in the neutrophil counts at 3 h after IL-6 injection. Clear expression of CD62L, CD11b, CD162, and IL-6R in neutrophils was observed in PBS-treated monkeys (Fig. 2B). CD162 expression was markedly reduced on neutrophils from IL-6-treated monkeys, but expressions of CD62L, CD11b, and IL-6R were not influenced (Fig. 2C). 3.3. IL-6 did not directly affect adhesion molecule expression on isolated human neutrophils We examined whether IL-6 directly reduced CD162 expression. Isolated human neutrophils were incubated with IL-6 for 2 h, and then CD11b, CD62L, and CD162 expression levels were analyzed. Human neutrophils also expressed IL-6R, and IL-6 induced cytokine production from human neutrophils (data not shown). Unexpectedly, IL-6 did not affect the expression of CD62L, CD11b, or CD162 on neutrophils at all (Fig. 3). 3.4. Involvement of GM-CSF and IL-8 in reduction of CD162 expression by IL-6 From the above results, we hypothesized that IL-6 indirectly reduced adhesion molecule expression via cytokine or chemokine production. To examine this idea, we measured plasma cytokine levels in IL-6-treated monkeys. Plasma concentration of IL-6 was increased by IL-6 injection and it was similar to that in CIA mon-
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Fig. 2. IL-6 increased neutrophil counts in blood and reduced adhesion molecule expression on neutrophils in monkeys. Monkeys were subcutaneously injected with cynomolgus monkey IL-6 (5 lg/kg) or PBS. Tocilizumab (8 mg/kg) was injected intravenously 24 h before IL-6 injection. At 3 h after IL-6 injection, peripheral blood was drawn. (A) Neutrophil counts in peripheral blood. (B) Representative expression levels of CD62L, CD11b, CD162, and IL-6R on neutrophils. Shaded histograms indicate the control IgG (isotype control); dotted lines indicate expression levels in a PBS-treated monkey; solid lines indicate expression levels in an IL-6-treated monkey. (C) Mean fluorescence intensities for CD11b, CD62L, CD162, and IL-6R. Each column and error bar indicates the mean and SE of three animals. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. PBS).
Fig. 3. IL-6 did not affect expression of adhesion molecules on human neutrophils. Human neutrophils were isolated from the peripheral blood of healthy subjects. Cells were incubated with IL-6 (10 ng/mL) for 2 h. After culture, expression of CD11b, CD62L, and CD162 on neutrophils was analyzed by the flow cytometry system. Data show the mean fluorescence intensities. Each column and error bar indicates the mean and SE of three donors.
keys as previously reported ( 300 pg/mL) [11]. Plasma concentrations of IL-8 and GM-CSF were increased by IL-6 injection, and tocilizumab inhibited IL-6-induced IL-8 and GM-CSF production (Fig. 4A). G-CSF expression was not induced by IL-6 injection (data not shown). On the other hand, tocilizumab increased blood IL-6 levels. Next, we examined the effect of IL-8 and GM-CSF on CD162 expression. As expected, IL-8 and GM-CSF significantly decreased CD162 expression on neutrophils (Fig. 4B). The combination of IL-8 and GM-CSF did not show a clear additive effect on the reduction of CD162 expression. The concentrations of the soluble form of CD162 were significantly higher in supernatants of neutrophils stimulated with GMCSF and IL-8 than in the supernatant from the non-treated group (Fig. 4C). We further studied whether a similar phenomenon would be observed in the case of human whole blood. Human whole blood was incubated with IL-6 for 2 h, followed by measurement of IL8 and GM-CSF in the cell supernatants. IL-8, but not GM-CSF, was induced by IL-6 (Fig. 5A). IL-6 also decreased CD162 expression
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Fig. 4. GM-CSF and IL-8 reduced CD162 expression on human neutrophils. (A) Serum levels of IL-8, GM-CSF and IL-6 in IL-6-treated monkeys were measured by using a Milliplex MAP Non-Human Primate Cytokine Panel. Each column and error bar indicates the mean and SE of three animals. Statistical significance was analyzed by unpaired ttest (p < 0.05 vs. PBS treated). (B) Human neutrophils were incubated for 2 h in the presence of IL-6 (10 ng/mL), GM-CSF (10 ng/mL), and IL-8 (10 ng/mL). After incubation, CD162 expression was measured by the flow cytometry system. Each column and error bar indicates the mean and SE of three donors. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. medium control). (C) Soluble CD162 levels in cell supernatants. Each column and error bar indicates the mean and SE of three donors. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. medium control).
Fig. 5. Human whole blood study. Human whole blood from three healthy subjects (500 lL) was incubated in a 24-well plate with IL-6 (10 ng/mL), tocilizumab (100 lg/mL), and anti-IL-8 antibody (500 lg/mL) for 2 h. After incubation, cells and plasma were collected. (A) The concentration of IL-8 in IL-6-treated whole blood. (B) Expression of CD162 on neutrophils. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. medium control).
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on neutrophils in whole blood, and the reduction of CD162 expression by IL-6 was reversed by tocilizumab and anti-IL-8 antibody, which restored CD162 expression to control levels (Fig. 5B). 3.5. GM-CSF and IL-8 induced expression of the cleavage enzyme ADAM10 that mediates CD162 shedding in isolated human neutrophils The fact that sCD162 increased in the supernatants of neutrophils stimulated with GM-CSF and IL-8 (Fig. 4C) suggested the cleavage of membrane-bound CD162 by a certain enzyme. ADAM8, ADAM10, and ADAM17 have all been shown to contribute to the shedding of adhesion molecules [18,19]. The role of ADAM proteins in CD162 shedding is not well characterized for any type of cell. To determine which ADAM family is involved in CD162 shedding, we examined ADAM8, ADAM10, and ADAM17 mRNA expression in neutrophils after stimulation by IL-6, IL-8, and GM-CSF. IL-8 and GM-CSF induced transcription of ADAM10, but not of ADAM8 or ADAM17, and IL-6 did not affect any of the ADAM8, ADAM10, or ADAM17 mRNA levels (Fig. 6A). 3.6. ADAM10 is involved in CD162 shedding in HL-60 cells To clarify the involvement of ADAM10 in CD162 shedding, we silenced ADAM10 mRNA with small interfering RNA (siRNA). In this study we used cells of the neutrophil-like cell line HL-60 because it takes several days to transfect siRNA into cells and isolated human neutrophils rapidly undergo apoptosis. HL-60 cells can be differentiated to a neutrophil-like phenotype by treatment with DMSO [17]. CD162 expression was induced in HL-60 cells treated with DMSO (HL-60D cells), compared with CD162 expression in HL-60 cells (Fig. 6B). GM-CSF and IL-8 induced ADAM10 protein in HL-60D cells, and ADAM10 siRNA inhibited induction of ADAM10 by GM-CSF and IL-8 (Fig. 6C). We further examined whether GM-CSF and IL-8 induced CD162 shedding. In control HL-60D cells, GM-CSF and IL-8 induced CD162 shedding like in isolated human neutrophils. On the other hand, in HL-60D cells with ADAM10 knocked-down, CD162 shedding was partially inhibited by GM-CSF and IL-8 (Fig. 6D). 4. Discussion The present report describes one mechanism of neutrophilia in arthritis. We showed that (1) IL-6 blockade rapidly improved neutrophilia in arthritic monkeys; (2) IL-6 injection increased neutrophil counts and remarkably reduced CD162 expression on circulating neutrophils in monkeys; (3) IL-6 injection induced GM-CSF and IL-8 production in monkeys; (4) GM-CSF and IL-8 reduced CD162 expression in human isolated neutrophils, but IL-6 itself did not; and (5) GM-CSF and IL-8 promoted shedding of CD162 in neutrophils via ADAM10 induction. These findings suggest that IL-6 is greatly involved in neutrophilia in arthritis. Moreover, IL-6 indirectly reduced expression of CD162 in neutrophils by inducing ADAM10 production from neutrophils. In this study, we showed that neutrophil counts were increased by collagen immunization and that high neutrophil counts were maintained after the onset of arthritis, and we showed that tocilizumab injection rapidly reduced the elevated neutrophil count to normal level. Moreover, elevated production of IL-6 is observed in the monkey CIA model [11]. These lines of evidence suggest that in this arthritis model, circulating neutrophils are increased by IL6. Tocilizumab reduces joint swelling and migration of neutrophils into inflamed joints [11], suggesting that a reduction in the number of blood neutrophils leads to improvement in joint swelling. Blood neutrophil counts are controlled by the differentiation and proliferation of precursor cells and by trafficking from the
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bone marrow and the marginal pool. It takes 7–10 days for myeloblastic and myelocytic progenitors to differentiate into neutrophils, but on the other hand, trafficking of neutrophils occurs rapidly [5]. IL-6 contributes to differentiation from progenitors to neutrophils [20]. Indeed, we previously reported that Gr-1 positive cells (neutrophils) in bone marrow and peripheral blood neutrophils significantly increase in IL-6 transgenic mice [21]. However, in this study IL-6 increased neutrophil counts very rapidly. These facts strongly suggest that IL-6 promoted the movement of neutrophils from the marginal pool to the blood. To the best of our knowledge, this is the first report demonstrating both in vivo and in vitro that IL-6 reduces CD162 expression on neutrophils. CD62L and CD162 are constitutively expressed on most circulating leukocytes, and they have been shown to play a role in leukocyte–endothelial interactions [22]. It is reported that IL-6 reduces CD62L expression on circulating neutrophils in rabbits [14]. However, as we also found in this study, it has been reported that changes in CD162 expression are more strongly correlated with neutrophil increase than changes in CD62L expression [23]. Isolated neutrophils expressed membrane-bound IL-6R, and IL6 induced IL-23 production from neutrophils (data not shown), demonstrating that neutrophils could respond to IL-6. However, IL-6 did not directly reduce CD162 expression on neutrophils. These results led us to hypothesize that cytokines or chemokines induced by IL-6 changed the expression of CD162. As expected, IL-6 injection induced IL-8 and GM-CSF production in monkeys, and these cytokines were found to be involved in reduction of CD162 expression on isolated neutrophils and in whole blood. However, it is necessary to perform further studies in order to conclude that IL-8 and GM-CSF reduces CD162 expression in vivo. It is reported that G-CSF also increases neutrophil counts and reduces CD162 expression on neutrophils in humans [23]. However, IL-6 did not increase serum G-CSF concentrations in monkeys. Therefore, the mechanism for the IL-6-induced decrease of CD162 expression might have no involvement with G-CSF. IL-6 injection induced GM-CSF and IL-8 production in monkeys. On the other hand, IL-6 induced IL-8 but not GM-CSF production from human peripheral whole blood in vitro. IL-8 and GM-CSF are produced by many cell types, including activated T and B cells, macrophages, monocytes, endothelial cells, and fibroblasts. We previously reported that IL-6 induces IL-8 in mononuclear cells of peripheral blood [9]. The difference between the in vivo and in vitro results may be explained as follows: IL-8 was induced by IL-6 from cells existing in circulating blood, but GM-CSF was from non-hematopoietic cells. In RA patients, serum concentrations of IL-6, IL-8, and GM-CSF are higher than those of healthy subjects [24]. Therefore, it is likely that IL-6 induces IL-8 and GM-CSF production in RA patients. GM-CSF and IL-8 decreased the membrane-bound CD162 and increased production of soluble CD162. This result strongly suggested that membrane-bound CD162 was cleaved. ADAM10 is expressed in most cells and plays the role of a secretase for numerous membrane proteins, including CD44, TNF, and E-cadherin [25]. It has been reported that ADAM10 is involved in CD162 shedding in monocytes [26]. In this study, we identified ADAM10 as a CD162 sheddase in neutrophils. However, because the influence of ADAM10 siRNA was partial, it is possible that another sheddase also mediates CD162 shedding from neutrophils. However, it is still unclear whether CD162 is essential for IL-6-induced neutrophilia. Further study should be performed to resolve this issue. Anti-IL-6R antibody (tocilizumab) treatment dramatically improves the symptoms of RA and normalizes neutrophil counts [27]. Our present study clearly suggests that over-production of IL-6 increases neutrophil counts in blood via the down-regulation of CD162 in RA patients, and that IL-6 blockade inhibits increased
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Fig. 6. ADAM10 mediated CD162 shedding from HL-60 cells. (A) Human neutrophils were incubated for 2 h in the presence of IL-6 (10 ng/mL), GM-CSF (10 ng/mL), and IL-8 (10 ng/mL). After incubation, mRNA levels of ADAM8, ADAM10, and ADAM17 were measured by real-time PCR. The ADAM expression by non-treated controls was defined as 1. Each column and error bar indicates the mean and SD of three donors. Statistical significance was analyzed by unpaired t-test (p < 0.05 vs. medium control). HL-60 cells were cultured with DMSO for 4 days and then cells were transfected with control siRNA and ADAM10 siRNA following the protocol of the transfection kit’s manufacturer. siRNA-treated cells were incubated with IL-8 (10 ng/mL) and GM-CSF (10 ng/mL) for 4 days. (B) CD162 expression in DMSO-treated HL-60 cells (HL-60D). Shaded histograms indicate non-treated HL-60 cells; solid lines indicate HL-60D cells. (C) ADAM10 expression of siRNA-treated HL-60D cells. ADAM10 and b-actin (internal control) protein expressions were measured by western blotting. Each column and error bar indicates the mean (n = 3) and SD. Statistical significance was analyzed by unpaired t-test (, #p < 0.05). (D) Effect of GM-CSF and IL-8 on CD162 in HL-60D cells. Shaded histograms indicate non-stimulated/control siRNA cells; solid lines indicate non-stimulated/ ADAM10 siRNA cells; thick lines indicate stimulated/control siRNA cells; dotted lines indicate stimulated/ADAM10 siRNA cells.
neutrophil counts induced by IL-6. Further studies in RA patients are warranted to confirm the results we obtained from the animal model.
In conclusion, IL-6 induces neutrophilia by promoting the production of IL-8 and GM-CSF which reduce expression of adhesion molecules on neutrophils.
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