ICAM-1 is a transmembrane glycoprotein of the Ig superfamily involved in cell adhesion. ICAM-1 is aberrantly expressed by astrocytes in CNS pathologies such as multiple sclerosis, experimental allergic encephalomyelitis, and Alzheimer’s disease, suggesting a possible role for ICAM-1 in these disorders. ICAM-1 has been shown to be important for leukocyte diapedesis through brain microvessels and subsequent binding to astrocytes. However, other functional roles for ICAM-1 expression on astrocytes have not been well elucidated. Therefore, we investigated the intracellular signals generated upon ICAM-1 engagement on astrocytes. ICAM-1 ligation by a mAb to rat ICAM-1 induced mRNA expression of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α. Examination of cytokine protein production revealed that ICAM-1 ligation results in IL-6 secretion by astrocytes, whereas IL-1β and IL-1α protein is expressed intracellularly in astrocytes. The involvement of mitogen-activated protein kinases (MAPKs) in ICAM-1-mediated cytokine expression in astrocytes was tested, as the MAPK extracellular signal-regulated kinase (ERK) was previously shown to be activated upon ICAM-1 engagement. Our results indicate that ERK1/ERK2, as well as p38 MAPK, are activated upon ligation of ICAM-1. Studies using pharmacological inhibitors demonstrate that both p38 MAPK and ERK1/2 are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 is important for IL-1α and IL-1β expression. Our data support the role of ICAM-1 on astrocytes as an inflammatory mediator in the CNS and also uncover a novel signal transduction pathway through p38 MAPK upon ICAM-1 ligation.

ICAM-1 is a transmembrane glycoprotein containing five Ig-like domains that is a member of the Ig superfamily (1). It is expressed on numerous cell types including leukocytes, endothelial cells, keratinocytes, and fibroblasts (for review, see Ref. 2). ICAM-1 serves as a ligand for the membrane-bound integrin receptors LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) on leukocytes (3). ICAM-1 plays an important role in leukocyte-endothelial cell interactions and leukocyte migration (for review, see Ref. 4). Interaction between ICAM-1 and LFA-1 on T cells is also involved in T cell activation and subsequent T cell-mediated immune responses (5, 6).

ICAM-1 is aberrantly expressed in the brain by glial cells in disorders such as multiple sclerosis (MS)3 and Alzheimer’s disease (AD) (Refs. 7, 8, 9 ; for review, see Ref. 10). In acute MS lesions, astrocytes are positive for ICAM-1 immunostaining, both within the lesion and also in the surrounding adjacent white matter beyond the lesion edge (11). In AD, reactive astrocytes at the periphery of neuritic plaques are positive for ICAM-1. Within such neuritic plaques, microglia express LFA-1, suggesting a possible role for ICAM-1/LFA-1-mediated interactions between astrocytes and microglia in AD (8).

The functional significance of ICAM-1 expression by astrocytes has not been well studied. Thus far, studies have mainly focused on its role in cellular adhesion between lymphocytes and astrocytes. Anti-ICAM-1 Ab inhibits leukocyte-astrocyte interactions in vitro (12). Similarly, anti-ICAM-1 Ab inhibits lymphocyte binding to tissue sections from the brains of experimental allergic encephalomyelitis-induced mice (13). It is also possible that ICAM-1 on astrocytes functions as a costimulatory molecule, considering the putative role of the astrocyte as an APC in the CNS (14, 15). The recent observation that ICAM-1 ligation on astrocytes induces TNF-α production (16) suggests that ICAM-1 may act as an “outside-in” signal transducer in these cells. The role of ICAM-1 as a signal transduction molecule has been studied using several different cell types, with varying results. In activated monocytes or T lymphocytes, ICAM-1 cross-linking inhibited the production of TNF-α, IL-1β, and IFN-γ (17). However, in a later study using a rheumatoid synovial cell line, cross-linking of ICAM-1 activated the transcription factor AP-1 and subsequently induced IL-1β transcription (18), suggesting cell-type specific “outside-in” signaling by ICAM-1. In peripheral blood monocytes, cross-linking of ICAM-1 facilitated the oxidative burst response (19). In A20 cells, a mouse B cell lymphoma cell line, ICAM-1 cross-linking up-regulated surface class II MHC molecule expression and also induced an increase in tyrosine phosphorylation of several cellular proteins including the Src family kinase, p53/p56lyn (20). ICAM-1 cross-linking induces tyrosine phosphorylation of the cytoskeletal-associated protein cortactin in brain microvessel endothelial cells, suggesting that signaling through ICAM-1 may affect cytoskeletal reorganization in these cells (21). These results collectively indicate that ICAM-1 cross-linking activates a diverse array of signal transduction cascades and biological functions, which may be cell-specific.

In this study, we have investigated the intracellular signals generated by ICAM-1 ligation in primary rat astrocytes. Our results indicate that ICAM-1 engagement induces expression of the cytokines IL-1α, IL-1β, IL-6, and TNF-α. Studies of the intracellular signaling pathways activated upon ICAM-1 ligation demonstrate the activation of p38 mitogen-activated protein kinase (MAPK) as well as extracellular signal-regulated kinase (ERK) 1/2 MAPK. In addition, both p38 and ERK1/2 MAPK are involved in ICAM-1-induced IL-6 expression, whereas only ERK1/2 MAPK participates in IL-1α and -1β expression. These results implicate ICAM-1 as functioning as an inflammatory mediator in the CNS through induction of cytokine expression.

Primary glial cell cultures were established from neonatal rat cerebra as described previously (22). Cells were cultured in DMEM, high glucose formula, supplemented with glucose to a final concentration of 6 g/L, 2 mM glutamine, 0.1 mM nonessential amino acid mixture, 0.1% gentamicin, 2.5 μg/ml amphotericin B, and 10% FBS (HyClone, Logan, UT). After 2 wk in primary culture, oligodendrocytes and microglia were removed by mechanical dislodgment. Astrocytes were harvested by trypsinization (0.25% trypsin, 0.02% EDTA) and monitored for purity by immunofluorescence. Astrocyte cultures were routinely >97% positive for glial fibrillary acidic protein, an intracellular Ag unique to astrocytes (23).

Mouse anti-rat ICAM-1 Ab (1A29) was purchased from Serotec (Raleigh, NC), polyclonal mouse IgG1 isotype Ab was obtained from Southern Biotechnology Associates (Birmingham, AL), and polyclonal rabbit anti-mouse IgG Ab (RAM) was purchased from Dako (Carpinteria, CA). Fab fragments of anti-ICAM-1 Ab were generated using the ImmunoPure Fab Preparation Kit from Pierce (Rockford, IL) according to manufacturer’s instructions. Purification of 100% pure Fab fragments was confirmed by Western blot assay. Rat recombinant TNF-α was purchased from BioSource International (Camarillo, CA). Goat anti-rat TNF-α neutralizing Ab, biotinylated anti-IL-1α Ab, and biotinylated anti-IL-1β Ab were obtained from R&D Systems (Minneapolis, MN). The p38 MAPK inhibitor SB202190 was purchased from Calbiochem (San Diego, CA) and the MEK inhibitor U0126 was obtained from Promega (Madison, WI). Phospho-ERK1/2 and phospho-p38 kinase Ab kits were purchased from New England Biolabs (Beverly, MA). Myelin basic protein (MBP) for the in vitro kinase assay was purchased from Sigma (St. Louis, MO). The rat cytokine template set rCK-1 was purchased from PharMingen (San Diego, CA).

Total cellular RNA was isolated from confluent monolayers of primary rat astrocytes that were incubated with anti-ICAM-1 Ab, Fab fragments of ICAM-1 Ab, or other stimuli as previously described (24). Briefly, cells were washed once in PBS and lysed directly in the culture dish. RNA was extracted with guanidinium isothiocyanate and phenol, and precipitated with ethanol. Ten micrograms of total cellular RNA was analyzed by RPA using an RPA kit (Ambion, Austin, TX) as previously described (25). Quantification of protected RNA fragments was performed by scanning with the PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and values for cytokine mRNA were normalized to GAPDH mRNA levels for each experimental condition.

Primary rat astrocytes were stimulated with medium alone, isotype or anti-ICAM-1 Ab, or TNF-α for various time periods, then culture supernatants were collected. As well, cell lysates were prepared from each sample by repetitive freeze-thaw cycles in 300 μl of TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Triton X-100). The expression of IL-1α and -1β protein was quantified using IL-1α and -1β ELISA kits (R&D Systems), respectively, according to the manufacturer’s instructions. Total protein concentrations from each cell lysate were measured using a Bio-Rad protein assay kit (Hercules, CA) and used for normalization.

IL-6 activity in astrocyte culture supernatants was determined in a biological assay using the IL-6-dependent B cell hybridoma B9 as previously described (26). Briefly, B9 cells (5 × 103 cells/well) were plated in 96-well microtiter plates, then serial dilutions of conditioned medium and recombinant human IL-6 (used as a standard) were added and incubated at 37°C for 72 h. Triplicate cultures were set up for each condition. After incubation, B9 cell growth was assessed using the MTT assay as described previously (26), and the amount of IL-6 in conditioned supernatants was determined by comparison to the recombinant IL-6 standard curve.

Control or anti-ICAM-1 Ab-stimulated cells were lysed using lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 25 μg/ml aprotinin, 25 μg/ml leupeptin), and protein concentrations were measured. Cell lysate (150 μg from each sample) was subjected to 15% SDS-PAGE. Proteins were then transferred to nitrocellulose membrane and probed with primary Ab (either biotinylated anti-IL-1α or -β Ab, or anti-phospho-ERK1/2). Enhanced chemiluminescence was used for detection of bound Ab. Membranes were stripped at 50°C for 1 h in buffer containing 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7, and were reprobed with primary Ab against ERK1/2 protein.

Astrocytes were cultured on glass slides and incubated with isotype control or anti-ICAM-1 Ab (5 μg/ml) for 24 h. Cells were fixed with 100% ethanol at room temperature for 5 min, washed three times with PBS, then incubated in a blocking solution containing 4% BSA, 10% normal goat serum, and 0.1% Triton X-100 in PBS for 30 min at room temperature. Samples were incubated with primary Ab (either isotype control or biotinylated-anti-IL-1β Ab; 0.2 μg/ml) in blocking solution for 1 h. After this, slides were washed and cells were incubated in 0.3% H2O2 for 15 min at room temperature to inactivate endogenous peroxidase; cells were then incubated with avidin-biotin-peroxidase complex for 1 h at room temperature. After a 15-min incubation in substrate solution (0.04% diaminobenzidine and 0.03% H2O2 in PBS), photographs were taken under a microscope using a Spot Digital Camera System (Diagnostic Instruments, Sterling Heights, MI).

Astrocytes were incubated with medium alone or anti-ICAM-1 Ab for various periods of time and lysed in 500 μl of lysis buffer. Soluble lysates (100–200 μg) were used to phosphorylate MBP (Sigma) in vitro as described (27). Lysates were incubated with 1 μg of anti-p38 MAPK Ab (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-ERK2 Ab (Santa Cruz) for 1 h at 4°C, followed by an overnight incubation with Protein A/G gel beads (Pierce, Rockford, IL). The immunocomplexes were washed four times in extraction buffer containing 1% Triton X-100 and twice in kinase reaction buffer (20 mM HEPES, pH 7.6, 0.1 mM vanadate, 20 mM MgCl2, 2 mM DTT, 20 mM β-glycerophosphate, 20 mM p-nitrophenyl phosphate). The washed immunocomplexes were incubated in 20 μl of kinase reaction buffer containing 10 μg of MBP and 5.0 μCi of [γ-32P]ATP for 20 min at 30°C. MBP phosphorylation was stopped by boiling in sample buffer followed by 10% SDS-PAGE and autoradiography.

Previous studies from our laboratory have shown that ICAM-1 is constitutively expressed on primary rat astrocytes (28). To investigate the putative involvement of ICAM-1 expression on astrocytes in inflammatory responses, we studied the effect of ICAM-1 ligation on the expression of cytokines. To initiate this study, mRNA expression of several proinflammatory cytokines in primary rat astrocytes upon ICAM-1 ligation was determined using a multiprobe RPA. As shown in Fig. 1,A, incubation of astrocytes with anti-rat ICAM-1 Ab (5 μg/ml) induced mRNA expression of IL-1α, -β, IL-6, and TNF-α by ∼15-, ∼40-, ∼20-, and ∼5-fold, respectively, at the 3-h time point (compare lanes 1 and 3). This level of induction was not further increased upon the addition of RAM, which cross-links the primary Ab (compare lanes 3 and 7). Incubation of cells with RAM alone had no influence on cytokine expression (compare lanes 1 and 5.) These data demonstrate that ICAM-1 binding to anti-ICAM-1 Ab is sufficient to induce proinflammatory signals. Next, astrocytes were incubated with various concentrations of anti-ICAM-1 Ab to determine the dose dependence of the cytokine induction (Fig. 1,B). IL-1α, IL-6, and TNF-α mRNA expression was induced at a concentration of 500 ng/ml, whereas IL-1β was induced at a lower concentration of 100 ng/ml. The induction level of all of the above cytokines peaked using 5 μg/ml of anti-ICAM-1 Ab (Fig. 1,B). Kinetic analysis studies demonstrate that mRNA expression of IL-1α, -β, and IL-6 peaks at 3 h of incubation with anti-ICAM-1 Ab (Fig. 1,C, lane 4) and returns to basal levels after 12 h. However, TNF-α is induced at an earlier time point; strong induction is observed at 1 h and peaks at 2 h (Fig. 1,C, lanes 2 and 3). To test whether IL-1 and/or IL-6 mRNA expression was due to TNF-α secreted upon ICAM-1 ligation, endogenously produced TNF-α was inhibited by the inclusion of anti-rat TNF-α-neutralizing Ab (Fig. 1,D). ICAM-1 ligation with anti-ICAM-1 Ab for 3 h induced IL-1α, -β, and IL-6 mRNA expression by ∼15-, ∼20-, and ∼30-fold, respectively (compare lanes 1 and 2). Expression of cytokine mRNA was not inhibited by the addition of anti-TNF-α-neutralizing Ab up to a concentration of 3 μg/ml (lanes 3–6). Interestingly, a partial inhibitory effect on ICAM-1-induced TNF-α mRNA expression was noted (lanes 5 and 6), suggesting autoinduction of TNF-α. Indeed, we have previously documented TNF-α induction of TNF-α gene expression in astrocytes (29). However, anti-TNF-α Ab at 3 μg/ml completely blocked TNF-α-induced cytokine expression (lanes 7 and 8), demonstrating that ICAM-1 ligation-induced IL-1 and -6 expression is not mediated indirectly by TNF-α production. A summary of the pattern of cytokine mRNA expression upon ICAM-1 ligation is shown in Table I. Please note that the fold induction range for TNF-α mRNA is large because its expression is variable.

FIGURE 1.

ICAM-1 ligation on rat astrocytes induces mRNA expression of proinflammatory cytokines. A, Primary rat astrocytes were incubated with medium alone (lane 1), mouse anti-rat ICAM-1 Ab (5 μg/ml) for 1–6 h (lanes 2–4), RAM (2.5 μg/ml) for 3 h (lane 5), or anti-ICAM-1 Ab (5 μg/ml) plus RAM (2.5 μg/ml) for 1–6 h (lanes 6–8). Total RNA was prepared from each sample and used for cytokine multiprobe RPA. The designation of each protected mRNA signal is denoted on the right. Data are representative of three independent experiments. B, Astrocytes were incubated with medium alone (lane 1) or anti-ICAM-1 Ab at varying concentrations for 3 h (lanes 2–6), then RNA was used for cytokine multiprobe RPA. Data are representative of two experiments. C, Cells were incubated with medium alone (lane 1) or anti-ICAM-1 Ab (5 μg/ml) for various time periods (lanes 2–7). RNA was prepared from each sample and used for cytokine multiprobe RPA. Data are representative of three experiments. D, Cells were incubated with medium alone (lane 1), anti-ICAM-1 Ab (5 μg/ml) for 3 h (lane 2), or various concentrations of anti-rat TNF-α Ab and anti-ICAM-1 Ab for 3 h (lanes 3–6). In separate experiments, astrocytes were stimulated with recombinant rat TNF-α (1 ng/ml) for 3 h (lane 7) or rat TNF-α (1 ng/ml) that had been incubated with anti-TNF-α Ab (3 μg/ml) for 1 h at room temperature before addition to the cells (lane 8). RNA was prepared and used for cytokine multiprobe RPA. Data are representative of two experiments.

FIGURE 1.

ICAM-1 ligation on rat astrocytes induces mRNA expression of proinflammatory cytokines. A, Primary rat astrocytes were incubated with medium alone (lane 1), mouse anti-rat ICAM-1 Ab (5 μg/ml) for 1–6 h (lanes 2–4), RAM (2.5 μg/ml) for 3 h (lane 5), or anti-ICAM-1 Ab (5 μg/ml) plus RAM (2.5 μg/ml) for 1–6 h (lanes 6–8). Total RNA was prepared from each sample and used for cytokine multiprobe RPA. The designation of each protected mRNA signal is denoted on the right. Data are representative of three independent experiments. B, Astrocytes were incubated with medium alone (lane 1) or anti-ICAM-1 Ab at varying concentrations for 3 h (lanes 2–6), then RNA was used for cytokine multiprobe RPA. Data are representative of two experiments. C, Cells were incubated with medium alone (lane 1) or anti-ICAM-1 Ab (5 μg/ml) for various time periods (lanes 2–7). RNA was prepared from each sample and used for cytokine multiprobe RPA. Data are representative of three experiments. D, Cells were incubated with medium alone (lane 1), anti-ICAM-1 Ab (5 μg/ml) for 3 h (lane 2), or various concentrations of anti-rat TNF-α Ab and anti-ICAM-1 Ab for 3 h (lanes 3–6). In separate experiments, astrocytes were stimulated with recombinant rat TNF-α (1 ng/ml) for 3 h (lane 7) or rat TNF-α (1 ng/ml) that had been incubated with anti-TNF-α Ab (3 μg/ml) for 1 h at room temperature before addition to the cells (lane 8). RNA was prepared and used for cytokine multiprobe RPA. Data are representative of two experiments.

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Table I.

Cytokine mRNA expression induced by ICAM-1 ligation

Optimal Time of Expression (h)Fold Inductiona
IL-1α ∼12–15 
IL-1β ∼25–40 
IL-6 ∼20–30 
TNF-α ∼5–30 
Optimal Time of Expression (h)Fold Inductiona
IL-1α ∼12–15 
IL-1β ∼25–40 
IL-6 ∼20–30 
TNF-α ∼5–30 
a

Compared to medium alone treatment. Range of fold induction from four experiments.

IL-1β protein expression upon ICAM-1 ligation was next examined. Astrocytes were incubated in medium alone (control), with isotype-matched Ab (5 μg/ml), or anti-ICAM-1 Ab (5 μg/ml) for 24 h, then supernatants were harvested and cell lysates were prepared from each sample as described in Materials and Methods. Secreted IL-1β protein was not detected in the supernatant from ICAM-1 Ab-activated cells (data not shown). IL-1β protein levels in the cell lysates of unstimulated or isotype control Ab-stimulated samples were also below the detection limit of the ELISA (Fig. 2,A). In contrast, IL-1β protein (42 pg/μg) was detected in cell lysates from ICAM-1 Ab-treated astrocytes (Fig. 2,A), demonstrating that ICAM-1 ligation induced intracellular IL-1β expression. It is known that IL-1β is expressed intracellularly as a proform, then later cleaved to the mature 17-kDa IL-1β species by the IL-1β converting enzyme (30). The m.w. of the IL-1β protein expressed in ICAM-1-ligated astrocytes was analyzed by Western blotting (Fig. 2,B). Upon ICAM-1 ligation, an anti-IL-1β Ab-positive band was detected at ∼31 kDa (compare lanes 1 and 2). This band correlated with that of a TNF-α-induced IL-1β proform (lane 3). It should be noted that TNF-α stimulation also led to the secretion of IL-1β protein (data not shown), which differs from our observations with ICAM-1 ligation. To localize the IL-1β proform induced by ICAM-1 ligation, immunostaining of rat astrocyte cultures was performed (Fig. 2, C and D). Astrocytes incubated with isotype control Ab were not stained with anti-IL-1β Ab (Fig. 2,C), whereas scattered IL-1β-positive astrocytes were detected in the ICAM-1 Ab-treated sample (Fig. 2,D). A higher magnification of the cells reveals that IL-1β localizes to the perinuclear region, as well as to the cytoplasm (Fig. 2 E). These results also indicate that not all astrocytes are capable of expressing IL-1β upon ICAM-1 ligation.

FIGURE 2.

IL-1β protein expression is induced upon ICAM-1 ligation on rat astrocytes. A, Astrocytes were incubated with medium alone, mouse IgG Ab (isotype Ab) (5 μg/ml), or anti-ICAM-1 Ab (5 μg/ml) for 24 h. Cells were lysed in TBST buffer, and 50 μl of cell lysate from each sample was used for the IL-1β ELISA. The amount of IL-1β expressed was normalized to the total protein concentration of each sample. Each experiment was performed in duplicate. Mean ± SEM of one representative experiment was shown from three independent experiments. B, Cells were treated with medium alone (lane 1), anti-ICAM-1 Ab (5 μg/ml) (lane 2), or TNF-α (50 ng/ml) (lane 3) for 24 h. Cell lysates were prepared and used for Western blot analysis. Cell lysate (150 μg) from each sample was resolved in 15% SDS-PAGE, transferred to nitrocellulose membrane, then immunoblotted with biotinylated anti-IL-1β Ab (0.2 μg/ml), followed by streptavidin-HRP complex (0.5 μg/ml). Left, Molecular mass marker of 31 kDa is shown. Representative of three experiments. C–E, Astrocytes were incubated with isotype-matched Ab (5 μg/ml) (C) or anti-ICAM-1 Ab (5 μg/ml) (D) for 24 h. Cells were fixed in 100% ethanol, incubated in blocking solution, and then incubated with biotinylated anti-IL-1β Ab (0.2 μg/ml) followed by avidin-biotin-peroxidase complex. E, IL-1β-positive astrocytes in higher magnification (×400).

FIGURE 2.

IL-1β protein expression is induced upon ICAM-1 ligation on rat astrocytes. A, Astrocytes were incubated with medium alone, mouse IgG Ab (isotype Ab) (5 μg/ml), or anti-ICAM-1 Ab (5 μg/ml) for 24 h. Cells were lysed in TBST buffer, and 50 μl of cell lysate from each sample was used for the IL-1β ELISA. The amount of IL-1β expressed was normalized to the total protein concentration of each sample. Each experiment was performed in duplicate. Mean ± SEM of one representative experiment was shown from three independent experiments. B, Cells were treated with medium alone (lane 1), anti-ICAM-1 Ab (5 μg/ml) (lane 2), or TNF-α (50 ng/ml) (lane 3) for 24 h. Cell lysates were prepared and used for Western blot analysis. Cell lysate (150 μg) from each sample was resolved in 15% SDS-PAGE, transferred to nitrocellulose membrane, then immunoblotted with biotinylated anti-IL-1β Ab (0.2 μg/ml), followed by streptavidin-HRP complex (0.5 μg/ml). Left, Molecular mass marker of 31 kDa is shown. Representative of three experiments. C–E, Astrocytes were incubated with isotype-matched Ab (5 μg/ml) (C) or anti-ICAM-1 Ab (5 μg/ml) (D) for 24 h. Cells were fixed in 100% ethanol, incubated in blocking solution, and then incubated with biotinylated anti-IL-1β Ab (0.2 μg/ml) followed by avidin-biotin-peroxidase complex. E, IL-1β-positive astrocytes in higher magnification (×400).

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Cell lysates as well as culture supernatants from unstimulated, isotype Ab- or ICAM-1 Ab-stimulated astrocytes (24-h incubation) were used for IL-1α detection by ELISA. Similar to the case of IL-1β, IL-1α protein was not detected in culture supernatants (data not shown). However, IL-1α protein expression was increased ∼5-fold in cell lysates from ICAM-1 Ab-treated astrocytes compared with that of untreated or isotype Ab-treated cells (Fig. 3,A). Western blot analysis was also performed using cell lysates; the IL-1α protein was barely detectable in control cell lysates (Fig. 3 B, lane 1), whereas a 31-kDa IL-1α-positive band was induced by ∼10-fold in ICAM-1 Ab-treated cell lysates (lanes 2 and 3). TNF-α stimulation for 24 h also induced the 31-kDa form of IL-1α (lanes 4 and 5). These data show that the 31-kDa putative proform of IL-1α is expressed intracellularly upon ICAM-1 ligation on astrocytes.

FIGURE 3.

IL-1α expression upon ICAM-1 ligation in astrocytes. A, Astrocytes were incubated with medium alone, mouse IgG Ab (isotype Ab) (5 μg/ml), or anti-ICAM-1 Ab (5 μg/ml) for 24 h. Cells were lysed in TBST buffer, and 50 μl of cell lysate from each sample was used for IL-1α ELISA. Each experiment was performed in duplicate, and mean ± SEM values are shown. Data are representative of two independent experiments. B, Astrocytes were incubated with medium alone (lane 1), anti-ICAM-1 Ab (5 μg/ml) (lanes 2 and 3), or TNF-α (50 ng/ml) (lanes 4 and 5) for 24 h. Cell lysates were prepared and used for Western blot analysis. Cell lysate (150 μg) from each sample was resolved in 15% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with biotinylated anti-IL-1α Ab (0.2 μg/ml) followed by streptavidin-HRP complex (0.5 μg/ml). Left, Molecular mass marker of 31 kDa is shown. Data are representative of two experiments.

FIGURE 3.

IL-1α expression upon ICAM-1 ligation in astrocytes. A, Astrocytes were incubated with medium alone, mouse IgG Ab (isotype Ab) (5 μg/ml), or anti-ICAM-1 Ab (5 μg/ml) for 24 h. Cells were lysed in TBST buffer, and 50 μl of cell lysate from each sample was used for IL-1α ELISA. Each experiment was performed in duplicate, and mean ± SEM values are shown. Data are representative of two independent experiments. B, Astrocytes were incubated with medium alone (lane 1), anti-ICAM-1 Ab (5 μg/ml) (lanes 2 and 3), or TNF-α (50 ng/ml) (lanes 4 and 5) for 24 h. Cell lysates were prepared and used for Western blot analysis. Cell lysate (150 μg) from each sample was resolved in 15% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with biotinylated anti-IL-1α Ab (0.2 μg/ml) followed by streptavidin-HRP complex (0.5 μg/ml). Left, Molecular mass marker of 31 kDa is shown. Data are representative of two experiments.

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IL-6 expression upon ICAM-1 ligation on astrocytes was next examined by measuring IL-6 bioactivity (Fig. 4). Astrocytes were treated with medium alone, isotype-matched Ab, or ICAM-1 Ab for 12 or 24 h, then supernatants were collected and used for the B9 bioassay, as described in Materials and Methods. Constitutive IL-6 protein expression was detected in supernatants from both unstimulated and isotype Ab-stimulated conditioned media (∼50–100 pg/ml of IL-6), and IL-6 protein levels were increased to ∼450 pg/ml (12 h) or ∼400 pg/ml (24 h) in supernatants from ICAM-1 Ab-treated astrocytes. These data demonstrate that ICAM-1 ligation induces IL-6 secretion by rat astrocytes.

FIGURE 4.

ICAM-1 ligation results in IL-6 secretion. Astrocytes were incubated with medium alone, isotype Ab (5 μg/ml), or anti-ICAM-1 Ab (5 μg/ml) for either 12 or 24 h. Supernatants were then harvested from each sample and used for the IL-6 bioassay as described in Materials and Methods. Mean ± SEM of one experiment assayed in triplicate is shown. Data are representative of four independent experiments.

FIGURE 4.

ICAM-1 ligation results in IL-6 secretion. Astrocytes were incubated with medium alone, isotype Ab (5 μg/ml), or anti-ICAM-1 Ab (5 μg/ml) for either 12 or 24 h. Supernatants were then harvested from each sample and used for the IL-6 bioassay as described in Materials and Methods. Mean ± SEM of one experiment assayed in triplicate is shown. Data are representative of four independent experiments.

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Our data show that the addition of anti-ICAM-1 Ab without any secondary Ab is sufficient to induce cytokine expression in rat astrocytes (Fig. 1,A). To test whether Ab binding to a single ICAM-1 molecule is sufficient to induce cytokine expression, we stimulated rat astrocytes with Fab fragments of anti-ICAM-1 Ab, then assessed cytokine mRNA expression by RPA (Fig. 5). Intact anti-ICAM-1 Ab (5 μg/ml) induced mRNA expression of IL-1α, IL-1β, IL-6, and TNF-α (lane 2) as previously shown. However, Fab fragments, at the concentrations of 1–5 μg/ml, failed to induce any of the cytokines above control levels (lanes 3–5). These data suggest that two molecules of ICAM-1 need to be cross-linked by divalent Ab to induce the ICAM-1-mediated inflammatory signal.

FIGURE 5.

Fab fragments of anti-ICAM-1 Ab do not induce cytokine mRNA expression. Astrocytes were incubated with medium alone (lane 1), anti-ICAM-1 Ab (5 μg/ml), or various concentrations of Fab fragments of anti-ICAM-1 Ab (1–5 μg/ml; lanes 3–5) for 3 h. Total RNA was prepared from each sample and used for cytokine multiprobe RPA. Data are representative of two independent experiments.

FIGURE 5.

Fab fragments of anti-ICAM-1 Ab do not induce cytokine mRNA expression. Astrocytes were incubated with medium alone (lane 1), anti-ICAM-1 Ab (5 μg/ml), or various concentrations of Fab fragments of anti-ICAM-1 Ab (1–5 μg/ml; lanes 3–5) for 3 h. Total RNA was prepared from each sample and used for cytokine multiprobe RPA. Data are representative of two independent experiments.

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As a next step, the possible signal transduction pathways involved in ICAM-1-mediated IL-1 and IL-6 expression were investigated. In particular, we focused on the involvement of ERK and p38 MAPK by using pharmacological inhibitors specific for ERK and p38 kinase signaling pathways. ICAM-1 induction of both IL-1α and -β was not affected by pretreatment of astrocytes with SB202190, a p38 kinase inhibitor (31), up to a 10 μM concentration (Fig. 6,A, lanes 3–6; Fig. 6, B and C). However, pretreatment of cells with U0126, a specific inhibitor of MAP kinase kinase (MEK) (32), inhibited ICAM-1 ligation-induced IL-1α and -β mRNA expression in a dose-dependent manner (lanes 7–10). U0126 at 10 μM inhibited IL-1α and -1β induction by ∼60 and ∼50%, respectively (Fig. 6, B and C). These data indicate that ICAM-1 ligation induces IL-1α and -β expression through an ERK signaling pathway, but does not involve the p38 kinase pathway. In contrast, IL-6 mRNA expression was inhibited by both p38 and MEK inhibitors (Fig. 6, A and D), suggesting that both p38 and ERK MAPK are involved in ICAM-1 ligation-mediated IL-6 expression. Furthermore, the two kinase inhibitors showed an additive inhibitory effect on ICAM-1-induced IL-6 expression (Fig. 6,A, lanes 11–14; Fig. 6 D).

FIGURE 6.

MEK1 and p38 MAPK inhibitors suppress ICAM-1-mediated cytokine expression. A, Astrocytes were incubated with medium alone (lane 1) or anti-ICAM-1 Ab (5 μg/ml) for 3 h in the absence (lane 2) or presence of the p38 kinase inhibitor, SB 202190 (lanes 3–6), the MEK1 inhibitor, U0126 (lanes 7–10), or both inhibitors (lanes 11–14) at various concentrations. SB 202190 or U0126 was added 30 min before the addition of anti-ICAM-1 Ab. Total RNA was prepared from each sample and used for cytokine multiprobe RPA. Data are representative of three independent experiments. B–D, The bands of each cytokine (IL-1α, IL-1β, and IL-6) and GAPDH mRNA were quantified using the PhosphorImager, and are shown as a percentage of the anti-ICAM-1-stimulated sample (set at 100%). The mean ± SEM of three independent experiments is shown. The difference between ICAM-1-treated and inhibitor-treated samples was significant as determined by the Student’s t test. ∗, p < 0.001; ∗∗, p < 0.02; n = 3.

FIGURE 6.

MEK1 and p38 MAPK inhibitors suppress ICAM-1-mediated cytokine expression. A, Astrocytes were incubated with medium alone (lane 1) or anti-ICAM-1 Ab (5 μg/ml) for 3 h in the absence (lane 2) or presence of the p38 kinase inhibitor, SB 202190 (lanes 3–6), the MEK1 inhibitor, U0126 (lanes 7–10), or both inhibitors (lanes 11–14) at various concentrations. SB 202190 or U0126 was added 30 min before the addition of anti-ICAM-1 Ab. Total RNA was prepared from each sample and used for cytokine multiprobe RPA. Data are representative of three independent experiments. B–D, The bands of each cytokine (IL-1α, IL-1β, and IL-6) and GAPDH mRNA were quantified using the PhosphorImager, and are shown as a percentage of the anti-ICAM-1-stimulated sample (set at 100%). The mean ± SEM of three independent experiments is shown. The difference between ICAM-1-treated and inhibitor-treated samples was significant as determined by the Student’s t test. ∗, p < 0.001; ∗∗, p < 0.02; n = 3.

Close modal

Based on the results obtained using the pharmacological inhibitors, we tested the direct activation of ERK, a substrate protein of MEK, upon ICAM-1 ligation on astrocytes. Using Abs recognizing phosphorylated ERK, we analyzed ERK1/2 activation by Western blot analysis (Fig. 7,A). In the absence of stimulation, constitutive expression of the phosphorylated forms of ERK1/2 are detected (Fig. 7,A, upper panel, lane 1). However, upon treatment with ICAM-1 Ab (10 μg/ml) for 1 h, levels of phosphorylated ERK1/2 increased up to 3-fold (lane 2). Incubation with RAM (5 μg/ml) did not affect the basal level of ERK1/2 phosphorylation (lane 3), nor did it further increase ICAM-1-induced phospho-ERK levels (lane 4). Stimulation of cells with TNF-α as a positive control showed similar levels of induction of phospho-ERKs (lane 5). The blot was stripped and reprobed with anti-ERK Ab, which recognizes total ERK1/2 protein (Fig. 7,A, lower panel), to demonstrate equivalent amounts of total ERK1/2 protein expression. We further confirmed ICAM-1-induced ERK activation using an in vitro kinase assay (Fig. 7,B). MBP was phosphorylated in vitro at a low level by incubation with ERK2 kinase from unstimulated astrocytes (Fig. 7,B, lane 1). However, the kinase activity of ERK2 rapidly increased upon ICAM-1 ligation; optimal activity was observed at 15 min, then returned to basal levels at 120 min (Fig. 7,B, lanes 2–6; Fig. 7,D). ICAM-1 ligation-induced p38 MAPK activation was also investigated using an in vitro kinase assay; p38 kinase activity slightly increased after 15 min and peaked at 30 min (∼3.5-fold induction) (Fig. 7, C and D). The levels of total p38 MAPK protein were consistent throughout these samples (data not shown). These data demonstrate that ICAM-1 ligation on astrocytes activates both p38 and ERK1/2 MAPK signaling pathways.

FIGURE 7.

ICAM-1 ligation activates ERK and p38 MAPK. A, Astrocytes were incubated with medium alone (lane 1), anti-ICAM-1 Ab (10 μg/ml) for 1 h (lane 2), RAM (5 μg/ml) for 30 min (lane 3), anti-ICAM-1 Ab for 1 h where RAM was added for the last 30 min (lane 4), or rat TNF-α (50 ng/ml) for 1 h (lane 5). Cell lysates were prepared, and then 40 μg of protein were electrophoresed in 12% SDS gels. Proteins were transferred to nitrocellulose membrane and probed with Ab specific to phosphorylated ERK1/ERK2 (0.5 μg/ml) (upper panel). The blot was stripped and reprobed with anti-ERK Ab for total ERK1/ERK2 protein levels (lower panel). Data are representative of three experiments. B, Astrocytes were incubated with anti-ICAM-1 Ab (5 μg/ml) for various time periods (0–120 min). Cell lysates were prepared from each sample, immunoprecipitated with anti-ERK2 Ab, then used for in vitro kinase assay using MBP as the substrate. After 20 min of the kinase reaction at 30°C, phosphorylated MBP was subjected to 12% SDS gel electrophoresis. The bands of phosphorylated MBP were quantified using the PhosphorImager and represented as fold induction in graph D, which was calculated by dividing the radioactivity of the anti-ICAM-1 Ab-stimulated samples by the activity of the unstimulated sample (0 h). Data are representative of two independent experiments. C, Astrocytes were incubated with anti-ICAM-1 Ab (5 μg/ml) for various time periods (0–60 min). Cell lysates were prepared from each sample, immunoprecipitated with anti-p38 MAPK Ab, then used for in vitro p38 kinase assay. The bands of phosphorylated MBP were quantified using the PhosphorImager and represented as fold induction in graph D. Data are representative of two independent experiments.

FIGURE 7.

ICAM-1 ligation activates ERK and p38 MAPK. A, Astrocytes were incubated with medium alone (lane 1), anti-ICAM-1 Ab (10 μg/ml) for 1 h (lane 2), RAM (5 μg/ml) for 30 min (lane 3), anti-ICAM-1 Ab for 1 h where RAM was added for the last 30 min (lane 4), or rat TNF-α (50 ng/ml) for 1 h (lane 5). Cell lysates were prepared, and then 40 μg of protein were electrophoresed in 12% SDS gels. Proteins were transferred to nitrocellulose membrane and probed with Ab specific to phosphorylated ERK1/ERK2 (0.5 μg/ml) (upper panel). The blot was stripped and reprobed with anti-ERK Ab for total ERK1/ERK2 protein levels (lower panel). Data are representative of three experiments. B, Astrocytes were incubated with anti-ICAM-1 Ab (5 μg/ml) for various time periods (0–120 min). Cell lysates were prepared from each sample, immunoprecipitated with anti-ERK2 Ab, then used for in vitro kinase assay using MBP as the substrate. After 20 min of the kinase reaction at 30°C, phosphorylated MBP was subjected to 12% SDS gel electrophoresis. The bands of phosphorylated MBP were quantified using the PhosphorImager and represented as fold induction in graph D, which was calculated by dividing the radioactivity of the anti-ICAM-1 Ab-stimulated samples by the activity of the unstimulated sample (0 h). Data are representative of two independent experiments. C, Astrocytes were incubated with anti-ICAM-1 Ab (5 μg/ml) for various time periods (0–60 min). Cell lysates were prepared from each sample, immunoprecipitated with anti-p38 MAPK Ab, then used for in vitro p38 kinase assay. The bands of phosphorylated MBP were quantified using the PhosphorImager and represented as fold induction in graph D. Data are representative of two independent experiments.

Close modal

We have investigated the effects of ICAM-1 ligation on inflammatory cytokine expression in primary rat astrocytes. Our results indicate that mRNA expression of IL-1α, IL-1β, and IL-6 is significantly up-regulated by ICAM-1 engagement (Fig. 1). As previously reported (16), TNF-α mRNA was also induced, although this result was not consistently observed. In brain microvessel endothelial cells, the ICAM-1-mediated signal can be amplified by the addition of secondary Ab (33). However, in astrocytes, the addition of secondary Ab (RAM) did not further enhance ICAM-1-induced cytokine mRNA expression (Fig. 1,A). In addition, Fab fragments of anti-ICAM-1 Ab did not induce cytokine expression (Fig. 5). These data suggest that the ICAM-1-mediated inflammatory signal is induced by ligation of two ICAM-1 molecules on rat astrocytes, which is distinct from that seen in endothelial cells (33). Kinetic analysis revealed that IL-1α, -β, and IL-6 mRNA expression was optimal at 3 h after ligation and returned to basal levels after 12 h, whereas TNF-α mRNA expression peaked 2 h after ligation. This observation raised the possibility that ICAM-1-induced IL-1 or IL-6 expression was mediated by TNF-α production. However, experiments using a TNF-α-neutralizing Ab demonstrated that ICAM-1-induced IL-1 or IL-6 production is independent of the action of TNF-α.

Interestingly, the results on cytokine protein production demonstrated that IL-1α and -β are expressed intracellularly, but are not secreted (Figs. 2 and 3). Both IL-1α and IL-1β are initially synthesized as 31-kDa proteins that are eventually proteolytically cleaved to generate the mature 17-kDa form (for review, see Ref. 34). Although the proform of IL-1β is regarded as inactive, the proform of IL-1α can exert similar functions as that of mature IL-1α (for review, see Ref. 34). Western blot analysis showed that most of the IL-1α and -β protein induced by ICAM-1 ligation exist as the 31-kDa proforms. Furthermore, the proform of IL-1β mostly localizes to the perinuclear region, although some staining throughout the cytoplasmic region was observed as well (Fig. 2 E). However, these data do not answer the question concerning the function of intracellular IL-1 expression in astrocytes, which remains to be elucidated in future studies. It is conceivable that these intracellular pools of IL-1 are cleaved and secreted upon a yet unidentified additional extracellular stimulus that leads to the activation of IL-1-converting proteases. In this regard, it is noteworthy that LPS-stimulated mouse macrophages produce large amounts of pro-IL-1β, which are secreted only after incubation with nigericin, a potassium ionophore (35). IL-1 has long been implicated in neurological diseases. Monocytes from MS patients express higher levels of IL-1α and -β than cells from normal controls (36), and IL-1-positive glial cells are easily detected by immunohistochemistry in MS brain tissue (37). Similarly, IL-1 has been implicated in AD (38); IL-1 can induce the expression of the Alzheimer amyloid-promoting factor antichymotrypsin in astrocytes (39) and plays a role in amyloid-β peptide secretion (40). In these studies, microglia are considered to be the major source of CNS IL-1, but our results suggest that IL-1 can also be expressed by astrocytes through ICAM-1-mediated signaling.

Unlike the case of IL-1, IL-6 is secreted from astrocytes upon ICAM-1 ligation (Fig. 4). The key role of IL-6 in CNS inflammation has been clearly demonstrated in IL-6 transgenic mice that constitutively express IL-6 under the control of the astrocyte-specific glial fibrillary acidic protein promoter (41). According to this report, IL-6 overexpression in the CNS causes severe neurologic disease with accompanying neurodegeneration, astrogliosis, and induction of several acute-phase proteins. IL-6 has also been reported to be up-regulated in several neurological diseases. For example, in MS patients, elevated levels of IL-6 are detected in cerebrospinal fluid in some studies (42). Similarly, IL-6 levels in cerebrospinal fluid are also elevated in AD patients (43). However, under physiological conditions, IL-6 has a neuroprotective role, supporting neuronal survival as well as neuronal regeneration (for review, see Ref. 44). Our previous studies demonstrated that IL-6 also has anti-inflammatory functions. For instance, IL-6 inhibits TNF-α expression (45) and cell adhesion molecule expression by astrocytes (46, 47). It is reasonable to propose that aberrant expression of ICAM-1 and its subsequent ligation on astrocytes may be partly responsible for the increased IL-6 levels in these diseases.

The intracellular signaling pathways involved in ICAM-1-mediated cytokine expression were also examined in this study. It has been previously reported that ICAM-1 ligation can induce ERK activation in human endothelial cells as well as in astrocytes (16, 48, 49). Based on these findings, we focused on the involvement of MAPK using pharmacological inhibitors. Our data show that ICAM-1-mediated induction of IL-1α and -β involves activation of ERK1/2, whereas activation of both ERK1/2 and p38 MAPK is required for IL-6 expression upon ICAM-1 ligation (Fig. 6). ICAM-1-induced p38 and ERK activation was demonstrated by an in vitro kinase activity assay (Fig. 7). To our knowledge, this is the first report that ICAM-1 ligation induces p38 MAPK activation in any cell type. Human ICAM-1, as well as rat ICAM-1, has a short cytoplasmic C-terminal tail (<30 aa) that is highly conserved (for review, see Ref. 2). Thus far, no protein interaction motifs have been identified in the ICAM-1 cytoplasmic region. A membrane-proximal 9-aa stretch (aa 478–486) containing a highly conserved tyrosine residue is involved in ICAM-1 interaction with the cytoskeletal protein α-actinin (50). However, no signaling molecule has been reported to interact with this 9-aa region or the rest of the cytoplasmic region. Therefore, it will be important to identify upstream signaling molecule(s) that interact with ICAM-1 and lead to the activation of MAP kinases and subsequent gene expression.

In conclusion, our study has demonstrated that ICAM-1 ligation on astrocytes can induce expression of proinflammatory cytokines (IL-1α, IL-1β, IL-6, and TNF-α). It is conceivable that within the diseased CNS parenchyma, ICAM-1-positive astrocytes interact with LFA-1- and/or Mac-1-positive cells (infiltrating inflammatory cells and resident microglia), leading to the production of proinflammatory cytokines by the astrocytes (for review, see Ref. 10). In addition, we have evidence that ICAM-1 ligation leads to induction of chemokine mRNA expression (IP-10, MCP-1, and MIP-1α) (data not shown). Aberrant chemokine expression is associated with the pathogenesis of many neurological diseases due to the chemoattractant properties of these mediators (for review, see Ref. 51). Thus, we propose that elevated expression of ICAM-1 on astrocytes during certain CNS diseases such as MS and AD can contribute to ongoing inflammatory responses by inducing the expression of proinflammatory cytokines and chemokines.

1

This work was supported in part by National Institutes of Health Grants MH55795, MH50421, and NS29719 (to E.N.B.). N.J.V. was supported by a National Institutes of Health Postdoctoral Fellowship (T32-AI07051).

3

Abbreviations used in this paper: MS, multiple sclerosis; AD, Alzheimer’s disease; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; RAM, rabbit anti-mouse IgG Ab; RPA, RNase protection assay.

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