Published online before print September 2, 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biochemistry, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University, Aachen, Germany
1 Correspondence: Department of Biochemistry, RWTH Aachen University, Pauwelsstrasse 30, 52072 Aachen, Germany. E-mail: schaper{at}rwth-aachen.de
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: IL-6 • inflammation • monocyte • cytokine
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
are the most prominent proinflammatory cytokines, whereas IL-10, TGF-β, and IL-17 are characterized by anti-inflammatory properties. The IL-6-type cytokines exert pro- as well as anti-inflammatory activities [1
]. IL-6 is the major activator of acute-phase protein expression in the liver, is a hematopoietic factor, and acts as a survival factor on neuronal cells. Furthermore, IL-6 is a differentiation factor for B and T cells and regulates cell proliferation. Whereas plasma cell proliferation is induced by IL-6, proliferation of melanoma cells is inhibited. Thus, the biological impact of the cytokine strongly depends on the target cell analyzed [1 ].
In the inflammatory reaction, the recruitment of leukocytes to the site of inflammation is a crucial step to cope with the noxa. Chemokines are canonical mediators of cell migration [2 ]. Initially, the potential of IL-6 to induce cell migration was attributed to IL-6-induced expression of the chemokines MCP-1 or IL-8 or adhesion molecules [3 4 5 ]. Recently, we presented evidence that IL-6, although not a classical chemokine, is also a rapid and direct mediator of T cell migration [6 ].
Whereas chemokines signal through seven-transmembrane receptors coupled to trimeric G-proteins, IL-6 acts through the single membrane-spanning receptor subunit gp130, which is associated with Janus kinases. IL-6 binding to the receptor complex, composed of two subunits gp130 and two subunits gp80 (IL-6R), leads to the activation of the JAK/STAT, MAPK, and PI-3K cascades [7
8
9
].
The most fundamental cellular requirements for cell migration are integrin activation followed by cell attachment and reorganization of the actin cytoskeleton [10 ]. In the study presented here, we analyzed the cellular activities of IL-6 involved in monocytic cell migration. We show β1-integrin activation, enhanced cell attachment to endothelial cells, actin polymerization, and fibronectin-dependent migration as well as transmigration through an endothelial cell layer in response to IL-6. Thus, IL-6 acts as a migration factor on monocytic cells and fulfills all biological properties to mediate cell migration. Our results further emphasize properties of IL-6 as a migration factor and will help to explain the proinflammatory potential of IL-6 by recruiting immune cells to the site of inflammation.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell culture
TUR cells were grown in RPMI (Gibco, Karlsruhe, Germany), supplemented with 10% FCS, streptomycin (100 mg/l), penicillin (60 mg/l), and G418 (400 mg/l). For migration assays, FCS and antibiotics were omitted from the medium. RAW 264.7 cells were grown in DMEM (Gibco), supplemented with 10% FCS, streptomycin (100 mg/l), and penicillin (60 mg/l). HUVEC and HMEC-1 were grown in endothelial cell growth medium MV (Promocell, Heidelberg, Germany), supplemented with 0.5% gentamycin.
Monitoring integrin activation
For monitoring activated β1-integrin, 5 x 105 TUR cells/ml were grown in RPMI supplemented with 2% FCS overnight and starved in 25 mM HEPES-buffer (pH 7.4), supplemented with 150 mM NaCl and 1 mM MgCl2 (or 5 mM MnCl2 for positive control of activated integrins). Stimulation was performed with 15 ng/ml IL-6 for the times indicated in the figures. The cells were incubated at 4°C for 30 min with 9EG7 antibodies, and after washing with modified FACS buffer [25 mM HEPES (pH 7.4), 150 mM NaCl, 5% FCS, 0.1% NaN3], rat IgG-specific, PE-conjugated goat antibodies were added. Unbound antibodies were eliminated by washing with modified FACS buffer. Subsequently, cells were monitored for activated β1-integrin subunits using a FACSCalibur (Becton Dickinson, Heidelberg, Germany).
Western blot analysis
Cells (5x106) were lysed in 500 µl lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA). Buffers were supplemented with aprotinin, pepstatin, and leupeptin (10 µg/ml each component). Cellular proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Antigens were detected by incubation with the appropriate primary antibodies and HRP-coupled secondary antibodies (1:5000; Dako, Hamburg, Germany). The membranes were visualized with an ECL kit (Amersham Biotech, Freiburg, Germany). Blots were stripped and reprobed to monitor equal loading of the gel.
Actin polymerization
For visualizing actin polymerization, 5 x 105 TUR cells/ml were grown in RPMI supplemented with 2% FCS, or 1 x 105 RAW 264.7 cells were grown in DMEM supplemented with 0.5% FCS overnight. Cells were starved in 25 mM HEPES buffer (pH 7.4), supplemented with 150 mM NaCl for 30 min, and subsequent stimulation was performed with 20 ng/ml IL-6 for the times indicated in the figure. Afterwards, the cells were fixed in 1.5% formaldehyde at room temperature for 10 min, permeabilized with 90% MeOH at 0°C for 10 min, and stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC; Fluka, Traufkirchen, Germany) at 4°C for 30 min. After washing with FACS buffer (PBS containing 5% FCS, 0.1% NaN3), the cells were monitored for F-actin using a FACSCalibur (Becton Dickinson).
Cell attachment assay
For cell-attachment assays, HUVECs were seeded into 96-well plates (6000 cell/well). After 24 h, the cells were stimulated with IL-1β (100 U/ml) for an additional 24 h to increase cell surface expression of cellular adhesion molecules. IL-1 was removed by washing the wells with RPMI medium. TUR cells were washed and resuspended in 25 mM HEPES buffer (pH 7.4)/150 mM NaCl in a final concentration of 1 x 107 cells/ml and subsequently left in the incubator for 10 min. Afterwards, the TUR cells were stimulated as indicated in the individual figure legends and dispended immediately for 5 x 105 cells/well. After 5 min incubation, attached TUR cells were fixed in 100 µl 5% glutaraldehyde, washed immediately 2x with PBS, and fixed again with 100 µl 5% glutaraldehyde at room temperature for 20 min. Afterwards, the attached cells were washed 3x with ddH2O and stained with crystal violet [0.1% (w/v) in 200 mM 2(-N-Morpholino)ethanesulfonic acid, pH 6.0] at room temperature for 60 min. After eliminating the excess of crystal violet by washing, the remaining dye was solubilized in 100 µl 10% (v/v) acetic acid for 5 min at 150 rpm on an orbital shaker. Absorbance of the probes was measured in a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm. The absorbance measured in each sample was corrected by the absorbance of samples without TUR cells [14
].
Isolation of primary CD14+ monocytes
For the isolation of primary PBMC, 20 ml Ficoll (GE Healthcare, Upsala, Schweden) was covered with 30 ml complete blood from healthy volunteers. After centrifugation (800 g) in a swing-out rotor at room temperature for 30 min, cells were taken from the interface and washed three times with MEM Eagle (Spinner modification, Sigma, Traufkirchen, Germany). The cells were resuspended at 1 x 108 PBMC/ml in PBS/0.1% BSA, supplemented with 2 mM EDTA.
CD14+ monocytes were isolated by use of the monocyte-negative isolation kit (Dynal, Karlsruhe, Germany).
Under agarose migration assays
Under agarose migration assays were performed according to the methods described by Nelson et al. [15
] and Foxman et al. [16
]. Agarose (2.4%) was suspended in Krebs Ringer HEPES buffer (130 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 10 mM HEPES, pH 7.4), boiled, and diluted with 1 vol Krebs Ringer HEPES buffer to yield 1.2% agarose. Solubilized, hot agarose was transferred to 35 mm tissue-culture dishes and incubated in a humidified incubator overnight. In the agarose, five wells in a row were prepared with a sterile plastic tip. The most peripheral wells (Wells 1 and 5) were filled with medium. Cells (105 in 10 µl medium) were added to the two intermediate wells (Wells 2 and 4). IL-6 (10 µl; 200 u/µl) was given into the central well (Well 3). Dishes were incubated overnight. Afterwards, cells were fixed with methanol for 30 min and then with 37% formaldehyde for an additional 30 min. Photos were taken from the left and right borders of the RAW 264.7 cells containing wells.
Modified Boyden chamber assays
For analyzing cell migration, 7.5 x 105 TUR cells/ml were incubated overnight in RPMI containing 2% FCS. Transwell inserts (6.5 mm diameter; 8 µm pore size; Corning Costar, Bodenheim, Germany) were coated with human fibronectin (5 µg/cm2). TUR cells (1x105)/100 µl cells were transferred into a Transwell and left for 30 min for sedimentation. The Transwell was inserted into a 24-well plate containing 800 µl medium. Cell migration to the bottom chamber was assessed 4 h later. For each experiment, the cells from four different fields were enumerated. Every migration assay was performed three times in triplicate. Migration was induced by addition of 20 ng/ml IL-6 into the lower compartment. The migration was expressed as means ± SEM.
Migration of isolated human primary CD14+ monocytes was analyzed with 1.6 x 105 cells transferred into fibronectin-coated Transwell inserts with 5 µm pore size.
For transmigration assays, instead of coating with fibronectin, 6000 HUVECs were seeded into the Transwell. After 24 h, the cells were stimulated with IL-1β (100 U/ml) for an additional 24 h to increase cell surface expression of cellular adhesion molecules. IL-1 was removed by washing the Transwells with RPMI medium.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
One initial step in cell migration is the regulation of integrin activity by chemokines, which controls attachment to cellular matrices or to cells. β1-Integrin is part of VLA-4 and binds to fibronectin or VCAM. 9EG7 antibodies against the active form of the β1-integrin can be used in FACS analyses to monitor cytokine-dependent β1-integrin activation [12 ]. RAW 264.7 macrophages were used as a model for adherent cells, whereas TUR cells were selected as a model for nonadherent cells. RAW 264.7 cells were stimulated with IL-6 for 2 and 30 min. As a positive control, the cells were incubated with manganese. Subsequently, β1-integrin activation was monitored by FACS analyses (Fig. 1 ). The experiments in Figure 1A show a rapid increase in β1-integrin activation in response to IL-6 and manganese, demonstrating that IL-6 is capable of activating β1-integrin in RAW 264.7 macrophages. To test whether the IL-6-mediated β1-integrin activation is really a result of IL-6, we added blocking antibodies against IL-6. The inhibitory function of the antibody was confirmed by impaired, IL-6-dependent STAT3 activation in the presence of the antibody (Fig. 1B) . Indeed, the IL-6-blocking antibodies eliminated IL-6-mediated β1-integrin activation. Additionally, we analyzed the integrin-activating potential of IL-6 on nonadherent monocytic TUR cells (Fig. 1C 1) . Figure 1C 2 shows that β1-integrin activation could already be observed after stimulation of TUR cells with IL-6 for 30 s.
 View larger version (34K): [in a new window] |
Figure 1. β1-Integrin activation in response to IL-6. (A) RAW 264.7 macrophages were stimulated with 15 ng/ml IL-6 for the times indicated in the individual histograms and stained for activated β1-integrin by FACS analysis using the 9EG7 antibody. Control analyses were performed in the absence of any stimulation (top left panel) and in the presence of Mn2+ (5 mM; top right panel). To control IL-6-mediated effects, blocking IL-6-antibodies (2 µg/ml) were added 30 min prior to stimulation with IL-6 (middle and bottom right panels). The histogram showing unstimulated cells in the top left panel (open histogram) was copied into the following panels (all others; filled histogram) for reference. The filled histogram in the top left panel represents fluorescence in the absence of the primary antibodies. (B) The IL-6-blocking antibody was controlled by analyzing IL-6-dependent STAT3 activation. RAW 264.7 cells were left untreated or stimulated with IL-6 for 5 or 20 min in the presence or absence of the antibody (2 µg/ml). Cellular lysates were prepared and analyzed for the tyrosine-phosphorylated (p)STAT3 and after stripping of the membrane for STAT3 protein to control equal loading of the gel. (C, 1) β1-Integrin activation after stimulation of TUR cells with 20 ng/ml IL-6 was analyzed, as described for RAW 264.7 cells in A. (C, 2) Short-term kinetics. The FACS analyses shown are representative of at least four independent experiments performed.
|
, IFN-
, and IL-1β to express adhesion molecules. Expression of VCAM-1 and ICAM1 was monitored by FACS (Fig. 2A
). IL-1β was found to be the most potent cytokine inducing ICAM-1 and VCAM-1 and therefore, used for priming in the following experiments. TUR cells were incubated with IL-6 or for positive control, with SDF-1 or manganese and subsequently, dispensed to the HUVEC-containing wells. Five minutes after the transfer, an attachment assay was performed as described in Materials and Methods. The amount of TUR cells attached to the layer of endothelial cell was determined by incorporation of crystal violet, corrected by the values taken from wells without TUR cells. Figure 2B
demonstrates enhanced attachment of TUR cells to a layer of endothelial HUVEC cells in the presence of IL-6, SDF-1, or manganese, indicating that IL-6 is competent to initiate monocyte attachment.
 View larger version (24K): [in a new window] |
Figure 2. IL-6-induced cell adhesion. (A) HUVECs were primed with TNF- (10 ng/ml), IFN- (1000 U/ml), or IL-1β (100 U/ml) for 24 h to compare the expression of adhesion molecules. Cell surface expression of ICAM-1 and VCAM-1 was monitored by FACS analyses. The filled histograms represent fluorescence in the absence of cytokines. One representative experiment out of three is shown. (B) The adhesion assays were performed on IL-1β-primed HUVECs. TUR cells were stimulated with IL-6 (20 ng/ml), stromal cell-derived factor 1 (SDF-1; 100 ng/ml), or Mn2+ (5 mM) for positive control and subsequently transferred to the multiwell plate containing the confluent layer of primed HUVEC cells, which were fixed, and nonadherent cells were eliminated by washing. Cells were stained with crystal violet, and their absorbance was measured at 570 nm and related to the cell numbers. The control represents adhesion of nonstimulated TUR cells (adhesion index=1). One representative adhesion assay out of three is shown. FL2-H, Fluorescence 2-height.
|
 View larger version (33K): [in a new window] |
Figure 3. Actin polymerization induced by IL-6. RAW 264.7 cells were stimulated with IL-6 (20 ng/ml) for the times indicated, and actin polymerization was monitored with TRITC-labeled phalloidin by FACS analyses (A) or immunfluorescence in the microsope (B). The histogram from the unstimulated cells in the top panel (open histogram) was copied into the following panels (filled histogram) for reference. The filled histogram in the top panel represents fluorescence in the absence of phalloidin. (B) Original bar, 10 µm (one representative experiment out of five or three, respectively). (C) TUR cells were analyzed as described for RAW 264.7 cells in A (one representative FACS analysis out of six is shown).
|
 View larger version (31K): [in a new window] |
Figure 4. Fibronectin-dependent migration in response to IL-6. (A) Migration of RAW 264.7 cells was analyzed in an "under agarose migration assay" as described in Materials and Methods. Two areas at the border of the RAW 264.7 cells containing wells are shown. The left pictures represent the border faced to the IL-6 reservoir, whereas the pictures on the right represent the border of the same cell-containing well but faced to a reservoir filled with medium alone. The RAW 264.7 cells preferentially migrate toward the IL-6 reservoir. The experiment has been repeated for more than five times, and the pictures shown are representative results. (B) Migration of TUR cells was analyzed in the presence or absence of fibronectin in a modified Boyden chamber assay. The membrane of the insert was coated with fibronectin or left untreated as indicated. IL-6 (20 ng/ml) was added into the lower compartment of the chamber. For control, no cytokine or SDF-1 (100 ng/ml) was added instead of IL-6. Migrating cells were counted 4 h later. Numbers were normalized to number of cells migrated in the absence of fibronectin and cytokines (migration index=1). (C) To discriminate chemokinesis from chemotaxis, the cytokine gradient was eliminated by adding IL-6 into the upper compartment. Migration under these conditions was monitored as described for B. One representative migration assay out of three or four is shown for B and C, respectively.
|
Furthermore, we performed experiments to discriminate between nondirected chemokinetic migration from directed chemotactic cell migration. Therefore, IL-6 was additionally added into the upper compartment to sequentially dissolve the cytokine gradient between the upper and the lower compartment of the Boyden chamber. Finally, the cytokine was added exclusively into the upper compartment. The results shown in Figure 4C indicate that the potential of TUR cells to migrate is reduced in parallel with the loss of the cytokine gradient. Administration of IL-6 into the upper compartment did not induce migration into the lower compartment of the Boyden chamber.
IL-6-induced chemotactic transmigration through a layer of endothelial cells
A crucial event in the inflammatory process is the migration of immune-competent cells to the site of inflammation. On this way, the cells have to leave the blood vessels and penetrate the layer of endothelial cells. Thus, we checked whether IL-6 is competent to induce transmigration of the TUR cells through a layer of endothelial HUVECs, which were seeded in noncoated Transwells to form a confluent layer of endothelial cells. As for the attachment assays, the cells were activated with IL-1β. TUR cells were distributed to the Transwells, and after sedimentation, IL-6 was added into the lower compartment of the Boyden chamber for 4 h to induce cell transmigration of the TUR cells through the layer of endothelial HUVECs (Fig. 5A left panel
). The number of TUR cells that migrated into the lower reservoir of the Boyden chamber was compared with the number of cells migrated in the absence of IL-6. This comparison indicates that IL-6 is competent to stimulate TUR cells to transmigrate through a layer of macrovascular endothelial cells. Similar observations were made for the migration of TUR cells through a layer of microvascular endothelial HMEC-1 cells (Fig. 5A right panel
).
 View larger version (19K): [in a new window] |
Figure 5. IL-6 induced transmigration through an endothelial cell layer. (A) Transmigration of TUR cells through a layer of endothelial cells was analyzed similarly, as described for the analysis of fibronectin-dependent migration in Figure 4B
. However, fibronectin was replaced by primed macrovasular endothelial HUVECs (left) or microvascular endothelial HMEC-1 (HMec). (B) To check for chemotaxic migration through a layer of HUVECs, the cytokine gradient was eliminated by adding IL-6 into the upper compartment as described for Figure 4C
. Migration under these conditions was monitored as described for A. One representative transmigration assay out of four is shown for both parts of the figure.
|
Integrin activation, actin polymerization, and migration of primary CD14+ human monocytes in response to IL-6 stimulation
To confirm our observation for primary monocytes, we isolated CD14+ monocytes from healthy volunteers. FACS analyses indicated that 93% of the isolated leukocytes were CD14+ cells (Fig. 6A
). Integrin activation on IL-6-stimulated CD14+ monocytes was monitored by FACS with the activation-specific β1-integrin antibody. Similarly, as shown for TUR and RAW 264.7 cells in Figure 1
, β1-integrin was activated by IL-6 on primary human CD14+ monocytes (Fig. 6B 1)
. Integrin activation by IL-6 could already be detected after 30 s of stimulation (Fig. 6B 2
; short-term kinetics). Furthermore, rapid actin polymerization, monitored by phalloidin incorporation, was observed in response to IL-6 (Fig. 6C)
. Most importantly, migration assays were performed in the absence of IL-6, in the presence of IL-6 in the lower compartment, and in the presence of IL-6 in both compartments. As already demonstrated for TUR cells, primary monocytes also migrate into the lower compartment, only in the presence of a cytokine gradient (Fig. 6D)
. In summary, these data confirm our data from cell lines and show that IL-6 induces chemotactic migration of primary human CD14+ monocytes.
 View larger version (28K): [in a new window] |
Figure 6. IL-6-induced integrin activation, actin polymerization, and migration of primary CD14-positive human monocytes. (A) CD14+ monocytes were isolated as described in Materials and Methods. The isolated leukocytes (93%) were positive for CD14, as determined by FACS analysis. (B, 1) CD14+ primary human monocytes were stimulated with 20 ng/ml IL-6 for the times indicated and analyzed for activated β1-integrin by FACS using the 9EG7 antibody. For control, analyses were performed in the absence of any stimulation (upper left panel) and in the presence of Mn2+ (5 mM; upper right panel). The histogram from the unstimulated cells in the upper left panel (open histogram) was copied into the following panels (filled histogram) for reference. The filled histogram in the upper left panel represents fluorescence in the absence of the primary antibodies. (B, 2) Short-term kinetics. Integrin activation is shown for one representative experiment out of three for both parts of the figure. (C) CD14+ monocytes were stimulated with IL-6 (20 ng/ml) for the times indicated, and actin polymerization was monitored with TRITC-labeled phalloidin by FACS analyses. The histogram from the unstimulated cells in the upper panel (open histogram) was copied into the following panels (filled histogram) for reference. The filled histogram in the upper panel represents fluorescence in the absence of phalloidin. One out of two experiments is shown. (D) Migration of CD14+ monocytes was analyzed in the absence of IL-6, in the presence of IL-6 (20 ng/ml) in the lower compartment, and in the presence of IL-6 (20 ng/ml) in both compartments to distinguish between chemokinesis and chemotaxis. One representative experiment out of three is shown.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The term "chemokine" is an artificial word to characterize the biological potential of chemotactic cytokines [17
]. Interestingly, this term is used exclusively for chemotactic cytokines signaling through G-protein-coupled receptors [2
]. Nevertheless, also, some cytokines signaling through receptor tyrosine kinases or receptor-associated kinases exert chemotactic activities. Migration in response to the growth factors platelet-derived growth factor and epidermal growth factor (EGF) has been studied in detail [18
]. Furthermore, IFN-2 has been demonstrated recently to induce leukocyte migration [19
].
IL-6 signals through a receptor complex composed of the single membrane spanning receptor subunits gp130 and IL-6R. Receptor activation results in the initiation of the JAK/STAT pathway and the MAPK and PI-3K cascades [9
, 20
]. The impact of IL-6 on leukocyte migration was demonstrated in IL-6 knockout mice, where recruitment of leukocytes to the site of inflammation is impaired [3
]. Previous studies focused on the potential of IL-6 to cause migration indirectly by inducing the expression and secretion of the classical chemokines MCP-1 or IL-8 by endothelial cells [5
, 21
]. Furthermore, IL-6, in concert with EGFR, was shown to induce migration of breast cancer cells through an autocrine mechanism [22
]. Only recently, migration as a direct response to IL-6 has been described for T cells [6
]. Until now, no detailed studies about the IL-6-induced cellular changes, which lead to migration, have been reported to confirm chemotactic activity of IL-6.
Here, we focused on biological activities leading to monocytic cell migration in response to IL-6. We show that IL-6 is competent to induce all biological changes in monocytes required for the transmigration from the blood vessel through the endothelium to the site of inflammation. In detail, we demonstrated that IL-6 activates β1-integrin (Fig. 1) , which as a component of the VLA-4 complex, is the ligand for fibronectin and VCAM-1 and thereby, could influence cell attachment. Indeed, increased attachment of nonadherent monocytic TUR cells on endothelial cells was detected in response to IL-6 (Fig. 2) . Migration of cells is characterized by actin turnover. Actin fibers are built up at the leading edge, whereas they are broken down at the trailing edge. In fact, actin polymerization and the assembly of F-actin in response to IL-6 were observed in the adherent RAW 264.7 macrophages, in nonadherent TUR cells, as well as in human primary monocytes (Figs. 3 and 6) . Finally, we showed fibronectin-dependent chemotactic migration (Figs. 4 and 6) and directed transmigration through a layer of endothelial cells (Fig. 5) . Thus, in summary, we were able to demonstrate that IL-6 fulfills all biological requirements to act as a chemotactic agent.
The lack of infiltrating monocytes in IL-6 knockout mice obviously confirms the relevance of IL-6 [23
]. However, the pitfall of an experimental system in vivo is that it is not possible to discriminate between indirect and direct effects of IL-6. The first leukocytes infiltrating the site of inflammation are neutrophils followed by monocytes [5
, 24
]. Activated neutrophils produce classical chemokines such as IL-8 but also IL-6, which induces IL-8 and MCP-1 expression in endothelial cells. As endothelial cells do not express the IL-6R, stimulation requires the presence of the agonistically acting, soluble (s)IL-6R, which is shed from apoptotic neutrophils [3
, 25
, 26
]. IL-6, together with the sIL-6R, could act via the induction of chemokine expression by endothelial cells and/or independent of the sIL-6R by direct induction of monocytic cell migration, as the IL-6R is expressed on the cell surface of monocytes.
In our well-defined in vitro system, we could present evidence that IL-6 is also able to directly induce monocytic cell migration even in the absence of neutrophils and endothelial cells on fibronectin-coated membranes. Worth mentioning, IL-6-induced transmigration, through a layer of endothelial cells (as all other experiments in this study), was analyzed without administration of sIL-6R. Furthermore, the requirement of an IL-6 gradient for cell migration argues against indirect effects of IL-6.
In summary, all of the data presented here give strong evidence for IL-6 as an additional, potent chemotactic agent for monocytic cell migration besides its already well-known effect on chemokine expression.
Received March 14, 2008; revised July 23, 2008; accepted August 11, 2008.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
6 β 1 Proc. Natl. Acad. Sci. USA 90,9051-90552 induces leukocyte integrin redistribution, increased adhesion, and migration J. Interferon Cytokine Res. 27,291-303[CrossRef][Medline] in serum and synovial fluid J. Rheumatol. 24,1510-1516[Medline]This article has been cited by other articles:
 |
|
 |
|
  H.-W. Koon, Y. S. Kim, H. Xu, A. Kumar, D. Zhao, I. Karagiannides, P. R. Dobner, and C. Pothoulakis Neurotensin induces IL-6 secretion in mouse preadipocytes and adipose tissues during 2,4,6,-trinitrobenzensulphonic acid-induced colitis PNAS, May 26, 2009; 106(21): 8766 - 8771. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. P. Christophi, C. A. Hudson, M. Panos, R. C. Gruber, and P. T. Massa Modulation of Macrophage Infiltration and Inflammatory Activity by the Phosphatase SHP-1 in Virus-Induced Demyelinating Disease J. Virol., January 15, 2009; 83(2): 522 - 539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Al-Banna, R. Raghupathy, and M. J. Albert Correlation of Proinflammatory and Anti-Inflammatory Cytokine Levels with Histopathological Changes in an Adult Mouse Lung Model of Campylobacter jejuni Infection Clin. Vaccine Immunol., December 1, 2008; 15(12): 1780 - 1787. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
