The Kinesin Superfamily Motor Protein KIF4 Is Associated With Immune Cell Activation in Idiopathic Inflammatory Myopathies (original) (raw)

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From the Neurology IV, Foundation Neurological Institute "Carlo Besta", Department of Medical Pharmacology, Milan, Italy (PB, CC, VN, FB, SR, PC, MM, LM, RM)

Send correspondence and reprint requests to: Pia Bernasconi, PhD, Neurology IV, Foundation Neurological Institute "Carlo Besta," Via Celoria 11, 20133 Milan, Italy; E-mail: pbernasconi@istituto-besta.it

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Cristina Cappelletti, PhD

From the Neurology IV, Foundation Neurological Institute "Carlo Besta", Department of Medical Pharmacology, Milan, Italy (PB, CC, VN, FB, SR, PC, MM, LM, RM)

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CNR Institute of Neuroscience, Cellular and Molecular Pharmacology Laboratory, Department of Medical Pharmacology, Milan, Italy (FN)

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From the Neurology IV, Foundation Neurological Institute "Carlo Besta", Department of Medical Pharmacology, Milan, Italy (PB, CC, VN, FB, SR, PC, MM, LM, RM)

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From the Neurology IV, Foundation Neurological Institute "Carlo Besta", Department of Medical Pharmacology, Milan, Italy (PB, CC, VN, FB, SR, PC, MM, LM, RM)

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ICREA and Centro de Regulación Genómica, Cell and Developmental Biology Program, Barcelona, Spain (IV)

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From the Neurology IV, Foundation Neurological Institute "Carlo Besta", Department of Medical Pharmacology, Milan, Italy (PB, CC, VN, FB, SR, PC, MM, LM, RM)

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From the Neurology IV, Foundation Neurological Institute "Carlo Besta", Department of Medical Pharmacology, Milan, Italy (PB, CC, VN, FB, SR, PC, MM, LM, RM)

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From the Neurology IV, Foundation Neurological Institute "Carlo Besta", Department of Medical Pharmacology, Milan, Italy (PB, CC, VN, FB, SR, PC, MM, LM, RM)

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From the Neurology IV, Foundation Neurological Institute "Carlo Besta", Department of Medical Pharmacology, Milan, Italy (PB, CC, VN, FB, SR, PC, MM, LM, RM)

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Supported by Grant Nos. RF2003.132 (to P.B.) and RF2002.159 (to R.M.) from the Italian Ministry of Health, Grant No. HPRN-CT-2000-0079 (to F.N.) from the European Union 5th Framework Program, Telethon Grant No. GTF05008 (to M.M.) from the Muscle Cell, Tissue, and DNA Biobank, and Grant No. 2005-2007 (to R.M.) from the Associazione Volontari Aiuti per la Sclerosi Multipla Italy.

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Pia Bernasconi, Cristina Cappelletti, Francesca Navone, Valeria Nessi, Fulvio Baggi, Isabelle Vernos, Stefania Romaggi, Paolo Confalonieri, Marina Mora, Lucia Morandi, Renato Mantegazza, The Kinesin Superfamily Motor Protein KIF4 Is Associated With Immune Cell Activation in Idiopathic Inflammatory Myopathies, Journal of Neuropathology & Experimental Neurology, Volume 67, Issue 6, June 2008, Pages 624–632, https://doi.org/10.1097/NEN.0b013e318177e5fd
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Abstract

The idiopathic inflammatory myopathies (IIMs) dermatomyositis, polymyositis, and inclusion body myositis are characterized by myofiber degeneration and inflammation. The triggering factors of muscle autoaggression in these disorders are unknown, but infiltrating T cells may be activated locally and proliferate in situ. T-cell polarization involving reorientation of cytoskeleton and microtubule-organizing centers mediated by motor proteins may occur within inflammatory cells in the muscle. We therefore analyzed ubiquitous and neuronal kinesin superfamily (KIF) members KIF-5, dynein, and KIF4 in IIM muscle biopsies and in activated peripheral blood lymphocytes from healthy donors. Only KIF-4 was altered. Transcript levels were significantly higher in IIM muscle than in controls, and KIF4+ inflammatory cells were found in IIM muscles. In polymyositis and inclusion body myositis, KIF4+ cells were mainly located around individual muscle fibers, whereas in dermatomyositis, they were also near blood vessels. KIF4+ cells were not specific to any immune lineage, and some were Ki67+. In peripheral blood lymphocytes stimulated with mitogens, interleukin 2 or anti-CD3/CD28 antibodies, KIF4 expression was upregulated, and the protein was localized in the cytoplasm in association with lysosome-associated membrane protein 1+ and perforin+ lysosomal vesicles. These results imply that KIF4 is associated with activated T cells, irrespective of their functional phenotype, and that it is likely involved in cytoskeletal modifications associated with in situ T-cell activation in IIM.

Introduction

Motor proteins of the kinesin superfamily (KIF) transport organelles, protein complexes, and mRNAs in a microtubule- and adenosine triphosphate-dependent manner from the Golgi apparatus to specific destinations within the cell (1). During mitosis and meiosis, KIFs are also involved in spindle organization and chromosome movements (2). In neurons, KIFs are essential for the anterograde transport of various molecules from the cell body to axons and dendrites, whereas the cytoplasmic dyneins are minus-end-directed microtubule motors that transport cargoes from axonal or dendritic terminals to the cell body (3).

One of the plus-end-directed microtubule motors, KIF4, was originally characterized as a mitotic motor implicated in chromosome segregation during mitosis (3). It has been reported, however, to be highly expressed in the developing nervous system and to colocalize with membrane organelles (4, 5). It seems that KIF4 plays a role in the microtubule-based transport of vesicles containing L1, a cell adhesion molecule, toward the tip of growth cones in developing neurons in culture (6).

Kinesin superfamily protein (KIF) 4 is absent or present at very low levels in most adult tissues: the notable exception is hematopoietic tissues in which KIF4 is abundantly transcribed and synthesized (4, 5). The Xenopus protein Xklp1, the homolog of human KIF4, is involved in chromosome segregation during mitosis (7), but it has recently been proposed to be involved in the regulation of microtubule dynamics (7, 8). The accumulating data on KIF4 location in adults and a possible role in microtubule dynamics suggested that it might be involved at certain stages of immune cell activation and/or proliferation. It is known that F-actin and actin-associated proteins are involved in establishing the interaction between the effector T cell and the target cell, but the detailed molecular mechanisms involved in the reorientation of the microtubule cytoskeleton and microtubule-based movement of the microtubule-organizing center to a point on or close to the cell surface, and the detachment of secretory lysosomes from the microtubules with subsequent docking to the lymphocyte membrane during maturation of the immunologic synapse, are poorly understood (9-13). As in other systems (14, 15), these processes are likely to require tight regulation of microtubule dynamics (16) and to involve the concerted action of various microtubule-stabilizing and/or microtubule-destabilizing proteins and microtubule-based molecular motors (3, 16).

The idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of subacute/chronic muscle disorders characterized by inflammation-mediated muscle injury and degeneration. The major forms of IIM are dermatomyositis (DM), polymyositis (PM), and sporadic inclusion body myositis (IBM) (17). Evidence supporting a primary autoimmune pathogenesis in PM and DM includes the presence of muscle damage at the endomysium (infiltrating T cells in PM, complement-mediated humoral attack against endothelial cells in DM), a frequent association with other autoimmune diseases, serum positivity for autoantibodies (e.g. anti-synthetase antibodies), and positive responses to immunosuppressive treatments (17). In sporadic IBM, it is unclear as to whether the immune response is primary or secondary (18). The triggering factors of myositis and the processes by which the immunologic attack induces muscle weakness are still unknown. Therefore, we investigated possible KIF involvement in immune cell activation by characterizing the expression of KIF4 and other kinesin motors in muscle biopsies from patients with IIM and in vitro during activation of peripheral blood lymphocytes (PBLs) from healthy donors.

Materials and Methods

Subjects

We analyzed muscle specimens from 39 patients with IIM (16 DM, 15 PM, and 8 IBM). Eight patients who had undergone diagnostic muscle biopsy but whose clinical, electromyographic, and histologic findings showed them to be free of muscle disease (hereafter called controls) were also included. All IIM patients met the diagnostic criteria for idiopathic DM, PM, or IBM (Table) (19, 20). No other neurologic or immunologic diseases were diagnosed. Muscle histopathology and inflammatory infiltrates were characterized by a routine diagnostic protocol (21). Consecutive transverse muscle sections, stained to reveal infiltrating cell phenotype (CD4, CD8, macrophages, and B-lymphocytes), direct cytotoxicity, and upregulation of major histocompatibility complex (MHC) Class I, were analyzed at 10× or 20× magnification as described elsewhere (21). Immunosuppressive drugs had been given to 2 patients only (1 DM and 1 PM) and to no controls prior to the muscle biopsy. In all cases, the biopsies had been obtained by needle biopsy, frozen in liquid nitrogen-cooled isopentane, and stored in liquid nitrogen. Written informed consent for muscle biopsy for diagnostic purposes and for tissue storage for research purposes was obtained from all patients as required by the ethical committee of the Foundation Neurological Institute.

TABLE.

Clinical Features and Immunopathologic Data of Inflammatory Myopathy Patients

TABLE.

Clinical Features and Immunopathologic Data of Inflammatory Myopathy Patients

Cells and Culture Conditions

For isolation of PBLs, heparinized venous blood from healthy volunteer donors was layered over Ficoll Hypaque Plus (Amersham Pharmacia Biotech AB, Piscataway, NJ) and centrifuged. The mononuclear cells thus obtained were washed twice with PBS (pH 7.2) and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum, 2 mmol/L of L-glutamine, 1% penicillin/streptomycin solution, 1% nonessential amino acids, 1 mmol/L of sodium pyruvate (all from Euroclone, Pero, Italy), and 5 × 10−3 mmol/L of 2-mercaptoethanol (BDH Biochemical, Poole, United Kingdom). Cells adherent to plastic dishes, after incubation for 2 hours at 37°C in 5% CO2, were eliminated. Peripheral blood lymphocytes were seeded at a concentration of 2 × 106 cells/ml in medium, supplemented as previously discussed, and incubated with various stimuli: concanavalin A (Con A; 1 μg/ml; Sigma, St. Louis, MO); recombinant human interleukin (IL) 2 (20 U/ml; Roche Diagnostics, Mannheim, Germany), plastic-immobilized anti-CD3 antibody (50-100 μl/well supernatant from clone TR66; American Type Culture Collection, Manassas, VA) alone or in combination with soluble anti-CD28 antibody (clone 37407; 5 mg/ml; R & D Systems, Abingdon, United Kingdom). Time courses of these experiments are reported in the Results section and figures. After incubation, the cells were harvested, centrifuged, and processed for RNA extraction and immunocytochemistry. Peripheral blood lymphocyte proliferation was assessed by pulsing cells with 1 μCi [3H]thymidine (Amersham Biosciences), and the incorporated radioactivity was determined after an additional 16 hours by liquid scintillation on a β-plate counter (MicroBeta TriLux, EG and G Wallac, Turku, Finland).

Neuroblastoma SH-SY5Y cells (ATCC CRL 2266; American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium High Glucose (Euroclone) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin solution and 1% L-glutamine (all from Euroclone) in a humidified atmosphere of 5% CO2 at 37°C. At confluence, the cells were harvested and processed for immunoblot.

Total RNA Extraction and cDNA Synthesis

Total RNA was extracted from 20 to 30 mg of frozen muscle tissue or 5 × 105 PBLs using TRIzol reagent (Invitrogen, Carlsbad, CA), followed by DNase I treatment (Ambion, Austin, TX). Random-primed cDNA was prepared using SuperScript II reverse transcriptase (Invitrogen) following the manufacturer's instructions and stored at −20°C pending polymerase chain reaction (PCR) amplification.

Real-Time Quantitative PCR

The TaqMan primer-probe combinations were designed and tested by Applied Biosystems (Foster City, CA). The assay codes were KIF4, Hs00602211_g1; ubiquitous KIF5, Hs00189659_m1; neuronal KIF5, Hs00189672_m1; and dynein, Hs00540753_m1. Each cDNA sample (corresponding to 100 ng total RNA) was amplified in triplicate using a 7500 Fast real-time PCR system (Applied Biosystems). Amplification of human glyceraldehyde-3-phosphate dehydrogenase (Applied Biosystems) served as endogenous control for sample normalization; normalized PCR amplification signals from nonmyopathic muscle cDNA or untreated PBLs served as calibrators. Transcript expression was quantitated by the ΔΔCT method according to the manufacturer's instructions (Applied Biosystems).

Protein Extraction

For protein extraction, 1 × 106 PBLs were seeded and maintained in culture for 72 hours in the absence or presence of Con A (1 μg/ml). At the end of this time, PBLs were collected, washed once with PBS, and resuspended in 120 μl of ice-cold RIPA lysis buffer (1% Nonidet P-40; 6.4 mmol/L of deoxycholate; 150 mmol/L of NaCl; 50 mmol/L of Tris-HCl, pH 7.5) containing protease inhibitors (1 mmol/L of sodium orthovanadate, 1 mmol/L of EGTA, 5 μg/ml of aprotinin, 12.5 mg/L of leupeptin, 1 mmol/L of phenylmethylsulfonyl fluoride) for 30 minutes (22). The lysates were passed through a 29-G needle and centrifuged for 10 minutes at 15,600 × g (IEC Micromax microcentrifuge) to pellet the nuclear fraction. The supernatant containing the cytosol fraction was moved to fresh tubes and used for immunoblot. The SH-SY5Y cell line was processed in the same way. Total protein concentrations were determined using the Coomassie Plus-The Better Bradford assay kit (Pierce, Rockford, IL).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Immunoblot

Aliquots (30 μg) of cytosol protein were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 4% stacking gel, and the gels were either stained with GelCode blue stain reagent (Pierce) or electroblotted to nitrocellulose membranes (Bio-Rad, Hercules, CA). Blots were incubated with rabbit polyclonal antibodies: anti-human ubiquitous kinesin heavy chain (23) (dilution, 1:1000), anti-human KIF-4 (C-tus), and anti-Xenopus KIF4 (Ab03; dilutions, 1:250) (24) overnight at 4°C. After incubation with goat anti-rabbit immunoglobulin G (whole molecule)-alkaline phosphatase (Sigma), blots were washed 3× 15 minutes in 20 mmol/L of Tris, pH 7.5, 150 mmol/L of NaCl, 0.1% Tween-20 plus 5% nonfat milk and 2 × 10 minutes in 20 mmol/L of Tris, pH 7.5, 150 mmol/L of NaCl. Immunoblot signals were developed using 1-step nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (Pierce), a 1-component sensitive precipitating alkaline phosphatase substrate. A PageRuler prestained protein ladder, a mixture of 10 recombinant, highly purified, colored proteins with apparent molecular weights of 10 to 170 kd (Fermentas, M-Medical, Florence, Italy), was loaded for monitoring protein separation, Western transfer efficiency, and protein sizes.

Immunohistochemistry

The following mouse anti-human monoclonal antibodies were used: anti-CD3, anti-CD4, anti-CD8, anti-CD22, anti-CD163, anti-MHC Class I, anti-MHC Class II (all from DAKO, Glostrup, Denmark), and anti-Ki67 (DAKO; kindly provided by Dr M. Barberis, MultiLab, Gruppo MultiMedica Milan, Italy). For KIF-4, rabbit anti-Xenopus polyclonal antibodies Ab03 and Ab65 (24) and the recently available affinity purified goat polyclonal antibody raised against a peptide mapping within an internal region of human KIF4A (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Cryostat consecutive transverse muscle sections (6-μm thick) were mounted on poly-lysine-coated glass slides (Bio-Optica, Milan, Italy), air-dried for 30 minutes at room temperature, and stored at −80°C until use. Frozen sections were fixed in acetone (−20°C) and rehydrated in PBS for 5 minutes. All staining was performed using an EnVision+System-HRP (3,3′-diaminobenzidine HCl+) kit (DAKO). After an endogenous peroxidase-blocking step and preincubation with protein block solution for 30 minutes, primary antibody at appropriate dilutions in PBS was added for 90 minutes, followed by washing in PBS and incubation (1 hour) with secondary antibody at room temperature. After 3 washes with PBS, the slides were stained by 5- to 10-minute incubation with the 3,3′-diaminobenzidine HCl+ chromogenic substrate system, which produces brown coloration at the antigen site. After extensive washes in distilled water, slides were counterstained with Mayer hematoxylin and mounted by Bio Mount (Bio-Optica).

Confocal Microscopy

Unfixed 6-μm-thick cryostat cut muscle sections and 4× 105 PBLs fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 were incubated with 10% normal goat serum diluted in PBS for 30 minutes at room temperature to block aspecific sites. Slides were then incubated with anti-KIF4, anti-perforin (Acris Antibodies, Hiddenhausen, Germany), anti-CD3, anti-CD8, anti-CD4, anti-CD163, anti-α-tubulin (Sigma), anti-lysosome-associated membrane protein 1 or anti-Ki67 primary antibodies and, after 3 washes with PBS, were treated with Cy2-conjugated goat anti-mouse immunoglobulin G and Cy3-conjugated goat anti-rabbit immunoglobulin G secondary antibodies (all from Jackson Immunoresearch Laboratories, West Grove, PA) for 1 hour. Fluorescence images were captured from an EclipseE600 confocal laser-scanning microscope (Nikon, Tokyo, Japan) and analyzed with LaserSharp 2000 software (Bio-Rad).

Statistical Analysis

Analysis of variance with post hoc Dunn multiple comparison was used to assess the significance of differences (considered significant when p < 0.05). Statview 5 for Macintosh (Abacus Concepts, Berkeley, CA) and GraphPad Prism version 4.0 for Macintosh (GraphPad Software, San Diego, CA) were used for data elaboration.

Results

Expression of KIF4 Transcript Is Upregulated in IIM Muscle Tissue

Kinesin superfamily protein (KIF) 4 transcript levels were assessed by real-time PCR in muscle tissue. Figure 1A shows that the expression of KIF4 mRNA was significantly higher in IIM than in control muscles (p < 0.001 for DM vs controls; p < 0.01 for IBM vs controls and PM vs controls). Kinesin superfamily protein (KIF) 4 mRNA levels in the 3 types of IIM were not significantly different. Variations in KIF4 transcript levels among patients with the same type of IIM were high (4- to 5,500-fold increases). To investigate whether increased mRNA levels in IIM were specific for KIF4, we assessed the expression of ubiquitous KIF5 (Fig. 1B) and of neuronal KIF5 (Fig. 1C) (23, 25). The mean levels of these transcripts were 2 to 3 times higher in IIM than in controls, but the difference was significant only for ubiquitous transcripts in IBM (mean, 3.6- ± 1.9-fold; p < 0.05; Fig. 1B). Cytoplasmic dynein transcripts (26) were also analyzed, but expression levels did not differ between IIM and controls (data not shown).

FIGURE 1.

Kinesin superfamily protein (KIF) 4 transcript expression is increased in dermatomyositis (DM), inclusion body myositis (IBM), and polymyositis (PM) muscle compared with control (CTRL). (A) Expression levels of KIF-4 mRNA as assessed by real-time polymerase chain reaction on muscle biopsies. Data were normalized by the level of glyceraldehyde-3-phosphate dehydrogenase expression in each sample and expressed as relative values by the 2−ΔΔCT method. Values from controls were used for calibration. Horizontal bars are means. (B) and (C) show levels of ubiquitous and neuronal KIF-5 mRNA, respectively. Levels are slightly increased (2- to 3-fold) in inflammatory myopathy muscles compared with control muscles.

KIF4 Protein Localizes in IIM Muscle-Infiltrating Immune Cells

To determine whether the increase in KIF4 mRNA in IIM muscle was accompanied by increased KIF4 protein and to identify cells expressing the protein, we used immunohistochemistry on serial sections of muscle. Kinesin superfamily protein 4 was not detected in control muscles, but there was intense KIF4 immunoreactivity associated with infiltrating mononuclear cells in IIM. The pattern of positivity in DM and PM muscles was closely similar to that of anti-CD3 (pan T cell)-, anti-CD22 (B cell)-, and anti-CD163 (macrophage)-positive cells (Fig. 2A). Similar results were obtained in IBM (not shown). Kinesin superfamily protein (KIF) 4 immunoreactivity was not associated with a specific subpopulation of mononuclear cells. In serial sections, CD3+ T cells (the most prominent immune cell population) seemed to be KIF4+, but CD22+ B cells and CD163+ macrophages may also have expressed KIF4 (Fig. 2A).

FIGURE 2.

Kinesin superfamily protein (KIF) 4 is localized in the immune cell-infiltrating muscle in patients with inflammatory myopathies. (A) Serial sections of dermatomyositis (DM), polymyositis (PM), and control (CTRL) muscle biopsies, immunostained to reveal KIF4, T lymphocytes (CD3), B lymphocytes (CD22), and macrophages (CD163). All are counterstained with Mayer hematoxylin. The pattern of KIF4 positivity is similar to that for CD3, CD22, and CD163 positivity, suggesting that KIF4 is expressed by infiltrating mononuclear cells but not preferentially expressed by any particular lineage. Kinesin superfamily protein 4 staining is not seen on muscle fibers. Kinesin superfamily protein (KIF) 4-positive cells surround or invade individual muscle fibers in PM or exit from blood vessels in DM (arrow). No KIF4 positivity is present in CTRL muscle. Original magnification: 40×. (A) Images represent 4 DM samples, 5 PM samples, and 3 CTRL samples. (B) Confocal microscopy of DM (upper panel) and PM (lower panel) muscle biopsies, representative of 2 DM and 2 PM samples stained. Kinesin superfamily protein (KIF) 4 is in red; CD4 (upper panel) and CD8 (lower panel) are in green. Colocalization is indicated by the merged images in yellow. Not all infiltrating mononuclear cells are KIF4+, and kinesin positivity is mainly at the site of contact of immune cells with muscle fibers. Original magnification: 40×.

In PM, which is characterized by cytotoxic damage to muscle fibers, KIF4+ cells were mainly located close to individual nonnecrotic muscle fibers, whereas in DM, KIF4 positivity was also found close to immune cells apparently exiting from blood vessels (Fig. 2A).

To establish whether KIF4 was indeed present on infiltrating T cells, we performed double-immunofluorescence confocal microscopy. We found that the protein colocalized with CD3+ T cells. The association was greater with CD4+ T cells in DM and with CD8+ T cells in PM in the restricted areas of muscle in which invading mononuclear cells were present (Fig. 2B). We also found that KIF4 was present on some CD163+ macrophages (data not shown).

Not all immune cells were KIF4+ because there was a lack of correlation between KIF4 mRNA levels and the numbers of infiltrating cells (Fig. 3A). In contrast, there was a strong correlation between KIF4 mRNA levels and the percentage of KIF4+ cells in the muscle infiltrates in all 3 IIMs (Fig. 3B).

FIGURE 3.

Kinesin superfamily protein (KIF) 4 mRNA levels correlate well with the percentage of cells positive for KIF4 protein. (A) Relative values of KIF4 mRNA, detected by real-time polymerase chain reaction (see Materials and Methods section and Fig. 1) in relation to total number of muscle-infiltrating cells, counted as described in Materials and Methods section. No correlation is evident. (B) Kinesin superfamily protein (KIF) 4 mRNA levels in relation to the percentage of KIF4+ cells in IIM muscle biopsies. The high levels of KIF4 transcript are associated with high proportions of cells positive for KIF4; low KIF4 transcript levels are associated with low proportions of KIF4+ cells (_R_2 = 0.78). The data points are representative values from biopsies of 2 DM, 1 PM, and 1 IBM patient.

Kinesin superfamily protein (KIF) 4 mRNA is not detected or present in low quantities in most nonproliferating adult tissues (4), but it is expressed at high levels in the thymus and spleen-the major sites of lymphocyte activation and proliferation (5). To investigate whether KIF4+ cells in IIM muscle were proliferating, serial sections of 2 DM, 2 PM, and 2 control muscle biopsies were processed by immunohistochemistry to reveal KIF4 and Ki67, a nuclear marker of proliferating cells (27). Although most infiltrating cells seemed to be KIF4+, only a proportion of these were Ki67+, indicating that a proportion was proliferating (Fig. 4). The proliferating cells were located close to muscle fibers.

FIGURE 4.

Kinesin superfamily protein (KIF) 4 is expressed in proliferating mononuclear cells identified by Ki67 expression on serial sections of dermatomyositis (DM), polymyositis (PM), and control (CTRL) muscle biopsies. The KIF4+ cells surround muscle fibers both in DM and PM. However, only a few Ki67+ cells are present in the adjacent section, indicating that a small proportion of the infiltrating mononuclear cells are proliferating. No staining for KIF4 or Ki67 is present on CTRL muscle sections. Original magnification: 40×. Images are representative of 2 DM samples, 2 PM samples, and 2 control samples.

Assessment of KIF4 mRNA and Protein Expression in In Vitro-Stimulated Immune Cells

To shed light on the factors that influence KIF-4 expression by immune cells in IIM, we investigated PBLs from healthy donors incubated with various stimuli. Kinesin superfamily protein 4 mRNA and protein levels were assayed as the cells proliferated in response to stimuli. Concanavalin A (1μg/ml) upregulated KIF-4 mRNA in a time-dependent manner (mean 4-fold increase ± 0.17 at 24 hours; 48-fold ± 0.09 at 72 hours) compared with basal levels (Fig. 5A). In contrast, ubiquitous KIF5 transcript levels were unaffected by Con A throughout the stimulation period (Fig. 5A). Enhanced KIF4 expression correlated with increased [3H] thymidine incorporation (_R_2 = 0.993; Fig. 5B).

FIGURE 5.

Upregulation of kinesin superfamily protein (KIF) 4 transcript correlates with cell activation and proliferation. (A) Peripheral blood lymphocytes were stimulated with mitogen concanavalin A (Con A) and harvested at 24, 48, and 72 hours and processed for real-time polymerase chain reaction (PCR) and [3H]thymidine incorporation. Transcriptional levels of KIF4 and ubiquitous KIF5 (uKIF5) are expressed as relative values and cell proliferation as counts per minute (cpm). Kinesin superfamily protein (KIF) 4 mRNA levels increase in parallel with [3H]thymidine incorporation during Con A stimulation, whereas uKIF-5 mRNA levels remain low throughout the stimulation period. (B) Strong correlation between cpm and KIF4 transcript levels (_R_2 = 0.993). (C) Peripheral blood lymphocytes were stimulated for 48 hours with Con A, 48 hours with IL-2 (which supports preferential growth and differentiation of T lymphocytes from a mixed cell population and stimulates lytic granule synthesis), and 6 days with plastic-immobilized anti-CD3 antibody alone or in combination with anti-CD28 antibody (T-cell receptor-mediated T activation). After incubation, the cells were harvested and processed for real-time PCR. The strongest activation signal (i.e. in the presence of both CD3 and CD28) is associated with greatest KIF4 expression. Data are means of 3 determinations and are representative of 3 independent experiments.

Peripheral blood lymphocytes were also incubated with IL-2 and anti-CD3 alone or in combination with anti-CD28. There was a mean 10-fold increase in KIF4 mRNA levels after 48 hours with IL-2, a mean 14-fold increase after 6 days with anti-CD3 alone, and a mean 42-fold increase with anti-CD3 and anti-CD28 costimulation (Fig. 5C). Increased expression of KIF4 mRNA was always associated with high cell proliferation.

Double immunofluorescence of IL-2-stimulated PBLs revealed that KIF4 was present mainly in the cytoplasm where it colocalized with lysosome-associated membrane protein 1 and, to a lesser extent, with perforin, indicating association with lytic granules (Fig. 6). Western blot analysis, using 2 KIF4-specific antibodies (Ab03 and C-tus), of low-speed supernatants (nuclei removed) from PBLs cultured for 24 hours revealed no KIF4 protein in unstimulated cells but a 50-kd band after Con A stimulation (Fig. 7). These results indicate increased KIF4 protein in Con A-stimulated cells, consistent with the transcriptional data shown in Figure 5. The same band was present in supernatants from SH-SY5Y human neuroblastoma cells. Ubiquitous KIF5 was always present in PBLs and neuroblastoma cells, but its levels did not change with Con A stimulation (Fig. 7).

FIGURE 6.

Intracellular distribution of kinesin superfamily protein (KIF) 4 after peripheral blood lymphocyte activation. Cells stimulated with interleukin 2 for 48 hours were processed for colocalization of KIF4 (red) with nuclear antigen Ki67, microtubule marker α-tubulin, cytolytic granule marker lysosome-associated membrane protein 1, and perforin (all green), and analyzed by confocal microscopy. After cell activation, KIF4 localizes in the cytoplasm (demonstrated by lack of colocalization with nuclear Ki67) near microtubules. In the tubulin panel, the white arrow indicates the microtubule-organizing center. Kinesin superfamily protein (KIF) 4 colocalizes in the cytoplasm with lytic granules, marked by lysosome-associated membrane protein 1 and containing perforin, when they were present. Original magnification: 60×.

FIGURE 7.

Kinesin superfamily protein (KIF) 4 protein levels are increased in activated peripheral blood lymphocytes (PBLs). Immunoblot of total cell extracts from PBLs unstimulated (−) or stimulated with 2 μg/ml concanavalin A (+Con A) for 72 hours. Ubiquitous KIF5 (uKIF5) was used as internal control, and undifferentiated SH-SY5Y (SY), a human-derived neurotypic cell line, was used as positive control for anti-KIF4 antibodies (Ab03 and C-tus). Left hand lane shows the prestained protein molecular weight marker. The blot is representative of 3 independent experiments.

Discussion

We have shown for the first time that the KIF4 motor protein (but not the other KIFs) is highly expressed in the muscle of patients with IIMs. Kinesin superfamily protein 4 transcripts were upregulated in IIM muscle compared with controls, but levels did not differ significantly between the 3 IIM forms. This indicates that upregulation is not specific for any of the IIM forms.

Serial section immunostaining indicated that the KIF4 protein was mainly associated with infiltrating mononuclear cells (T lymphocytes, B lymphocytes, and macrophages) surrounding muscle fibers. Confocal microscopy showed that the KIF4 protein was associated with CD4+ T lymphocytes in DM and CD8+ T lymphocytes in PM (Fig. 2), and also with some CD163+ macrophages. Not all immune cells were KIF4 positive, however, as was also indicated by the lack of correlation between KIF4 mRNA levels and numbers of infiltrating cells (Fig. 3A). This suggested to us that KIF4 might be present in the cells only at certain stages of their life cycle or function.

We first sought to determine whether KIF4 positivity correlated with proliferation and found in agreement with Lindberg et al (28) that some KIF4-positive cells in IIM muscles also expressed Ki67 (Fig. 4), a marker of proliferating cells. The association KIF4-Ki67 is consistent with the idea that KIF4 is involved in immune cell proliferation in muscle in vivo. This concept is further supported by our finding that many stimulated PBLs expressed both KIF4 and Ki67 proteins (Fig. 6). Furthermore, Con A-stimulated PBLs showed markedly increased expression of KIF4 transcripts that correlated with [3H]thymidine incorporation (Fig. 5B), but no increase of ubiquitous or conventional KIF5 transcripts, indicating that KIF4 expression in mononuclear cells is a specifically inducible process. The results of our Western blot analysis of PBLs with 2 different KIF4-specific antibodies demonstrated that KIF4 was present only in stimulated (Con A) cells (Fig. 7); moreover, it was present in the cellular fraction corresponding to the cytoplasm. These antibodies also recognized a band with identical electrophoretic mobility in the neuroblastoma cell line, demonstrating that the protein expressed in PBLs was the same as that expressed in neuronal cells.

In all PBL experiments, KIF4 expression continued to increase throughout the stimulation period (up to 6 days in the case of CD3/CD28), with no decline observed. By analogy with our PBL data, the KIF4-positive muscle-infiltrating cells are also likely to be activated. This might occur as a result of T-cell interaction with the peptide-MHC complex on antigen-presenting cells; dendritic cells are known to be present in IIM infiltrates (29) or on muscle fibers. This activation can also occur as a consequence of interaction of the T-cell ligands CD28/CTLA4 and CD40L with CD80 or CD40, respectively, the costimulatory molecules expressed on antigen-presenting cells (29, 30). It might also be triggered by soluble factors. Indeed, many cytokines, predominantly of the TH1 type, have been reported in IIM muscles. Infiltrating cells, muscle fibers, and endothelial cells are potent producers of proinflammatory cytokines, including IL-1, IL-6, IL-2, and IL-18 (31-33).

The fact that perforin and granzyme transcripts were present in IIM muscle (34, 35) also suggests activation because these transcripts are normally present in T cells only after CD3/CD28 engagement (36, 37). We also found that perforin expression in PBLs increased after CD3/CD28 and IL-2 stimulation. All these considerations suggest that KIF4 might be associated with lytic granule transport. Indeed, we found in PBLs that KIF4 colocalized with lysosome-associated membrane protein 1, marker of lytic granules, and also, to a considerable extent with perforin, which is normally present in the granules (Fig. 6). Thus, KIF4 may be associated with these granules. Stinchcombe et al (12) have suggested that granule delivery to the lytic synapse might be driven by reorganization of the actin and microtubule cytoskeleton, with clearing of the plus-ends of microtubules away from the area of contact between T cell and antigen-presenting cells. In light of these data, we suggest that KIF4, one of the plus-end-directed microtubule motor proteins, may contribute to the structural rearrangement of the cytoskeleton necessary for granule dissociation and release. This suggestion is consistent with recent findings that KIF4 in undifferentiated neurons (Navone et al, unpublished results), and the KIF4 homolog of Xenopus (7, 8) affect microtubule polymerization and stability. Our finding that KIF4 is specifically upregulated in infiltrating cells in IIM muscle leads us to hypothesize that KIF4 may be a marker of cell activation, although this requires confirmation by studies on other inflammatory (immune or autoimmune) conditions.

In conclusion, we have shown for the first time that the KIF4 motor protein is present in the muscles of patients with IIM and also in activated PBLs in vitro. Although our experimental approaches have not enabled us to elucidate the full biologic role(s) of KIF4 in the inflammatory process, they imply that KIF4 is associated with activated T cells, irrespective of their functional phenotype. Therefore, KIF4 is likely to be involved in the cytoskeleton modifications associated with T-cell activation. Clearly, further studies are required to elucidate the role of this motor protein in IIM and other inflammatory processes.

Acknowledgments

The authors thank Dr M. Barberis (MultiLab) for providing the anti-Ki67 antibody and Don Ward for help with the English.

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Author notes

Supported by Grant Nos. RF2003.132 (to P.B.) and RF2002.159 (to R.M.) from the Italian Ministry of Health, Grant No. HPRN-CT-2000-0079 (to F.N.) from the European Union 5th Framework Program, Telethon Grant No. GTF05008 (to M.M.) from the Muscle Cell, Tissue, and DNA Biobank, and Grant No. 2005-2007 (to R.M.) from the Associazione Volontari Aiuti per la Sclerosi Multipla Italy.

Copyright © 2008 by the American Association of Neuropathologists, Inc.

Topic:

• phenotype
• polymyositis
• dermatomyositis
• inflammatory cells
• inflammation
• aldesleukin
• biopsy
• cd28 antigens
• cytoplasm
• cytoskeleton
• dynein atpase
• ki-67 antigen
• kinesin
• lymphocytes
• lysosomes
• membrane proteins
• microtubule-organizing center
• mitogens
• muscle fibers
• myositis, inclusion body
• rna, messenger
• t-lymphocytes
• idiopathic inflammatory myopathy
• antibodies
• vesicle
• immune cell activation
• t-cell activation
• molecular motor proteins
• perforin
• tissue degeneration
• donors

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