Interleukin-1β Induces Tissue- and Cell Type–Specific Expression of Adhesion Molecules In Vivo

Reagents

Dulbecco’s PBS was purchased from GIBCO-BRL. Agarose, diethylpyrocarbonate, dextran sulfate, and MOPS were from Sigma Chemical Co. Nick translation kits and proteinase K were purchased from Boehringer Mannheim Yamanouchi. Formamide and 50× Denhardt’s solution were obtained from Wako Pure Chemical Industries Inc. cDNA synthesis kits were purchased from Pharmacia LKB Biotechnology. In vitro transcription reagents were obtained from International Biotechnology. RNase-free DNase and RNase inhibitor were obtained from Promega. Restriction endonucleases and T4 polynucleotide kinase were purchased from Takara Shuzo Co. Paraformaldehyde was from Taab Laboratories Equipment Ltd. Dupont–NEN Research Products supplied [α-³²P]dCTP, [γ-³²P]ATP, ³⁵S-CTP, and ¹²⁵I–recombinant IL-1β. Recombinant IL-1β (5×10⁸ U/mg) and TNF-α (4×10⁷ U/mg) were from Genzyme Corp. LPS (prepared by Westphal phenolic extraction from Escherichia coli 026:B6) was purchased from DIFCO Laboratories. All other chemicals were obtained from Nacalai Tesque Inc.

Mice

Specific pathogen–free, inbred C57BL/6 mice, 9 to 12 weeks of age, were purchased from Charles River Japan Inc (Yokohama, Japan). Animals were maintained in microisolation cages with autoclaved food and bedding to minimize exposure to viral and microbial pathogens.

Experimental Design

Mice were injected with IL-1β, TNF-α, or PBS (vehicle) into the tail vein as indicated in the text. At the indicated times, the mice were killed, and their organs were removed, minced into small fragments, and homogenized in guanidine isothiocyanate solution before preparation of total RNA as described below. For in situ hybridization studies, 1 hour after injection of IL-1β (50 ng per mouse) or TNF-α (500 ng per mouse), the mice were anesthetized with ether and were fixed in toto by cardiac perfusion with 4% buffered paraformaldehyde solution. Hearts and lungs were removed, excised, and immersed overnight in the same fixation buffer. All animal experiments were conducted in accordance with the Guidelines for Animal Experiments of J.T. Pharmaceutical Research Laboratories.

Preparation of Plasmid DNA and Oligonucleotide Probes

Fragments of murine E-selectin,¹⁴ ICAM-1,¹⁸ and VCAM-1¹⁹ genes were obtained by reverse transcription–polymerase chain reaction by using the template cDNAs prepared from total RNA extracted from the hearts of IL-1β–treated mice. The fragments were cut by restriction enzymes and inserted into pBluescript II (Stratagene). Fragments were of the following sizes: 570 bp for murine ICAM-1, 360 bp for VCAM-1, and 770 bp for E-selectin. Plasmid DNA (1 μg) was radiolabeled by nick translation with [α-³²P]dCTP to a specific activity of ≈10⁸ counts per minute per microgram. A synthetic oligonucleotide corresponding to part of the rat α-tubulin gene was purchased from Oncogene Science Inc. The oligonucleotide (10 pmol) was radiolabeled by T4 polynucleotide kinase with [γ-³²P]ATP to a specific activity of >10⁶ cpm/pmol. Each probe was used at 7×10⁶ cpm per blot.

Preparation of RNA and Northern Blot Hybridization

Total RNA was extracted from ≈0.7 to 1 g of each organ removed after intravenous treatment by homogenization with a Polytron sonicator/homogenizer for 2 minutes in guanidine isothiocyanate–CsCl according to previously published methods.²⁰

Equal amounts of RNA (20 μg) were denatured and subjected to electrophoresis in a 1% agarose-formaldehyde gel. The RNA was then blotted by capillary transfer onto nylon membranes (Boehringer Mannheim Yamanouchi). The blots were prehybridized for 6 hours at 42°C in 50% formamide, 1% SDS, 5× SSC, 1× Denhardt’s solution (0.02% Ficoll, 0.02% BSA, and 0.02% polyvinylpyrrolidone), 0.25 mg/mL denatured herring testis DNA, and 50 mmol/L sodium phosphate buffer, pH 6.5. Hybridization was carried out at 42°C for 12 to 18 hours with 7×10⁶ cpm of denatured probe. The filters were washed for 30 minutes at room temperature in 0.1% SDS–2× SSC for 15 to 30 minutes at 55°C in the same solution. The blots were then exposed to XAR-5 x-ray film (Eastman Kodak) with Dupont Cronex Lightening Plus intensifying screens at −70°C. In some experiments, the blots were reused by stripping and rehybridizing with different probes. Expression of α-tubulin was used as an internal control in all experiments. In addition, the RNA load per lane was assessed by ethidium bromide staining of the original agarose gel after capillary transfer.

In Situ Hybridization

In situ hybridization was performed using the method described by Stoler.²¹ Antisense and sense radiolabeled ³⁵S-cRNA probes for E-selectin, ICAM-1, and VCAM-1 were prepared by in vitro transcription from T3 and T7 RNA polymerase promoters, respectively, in the pBluescript plasmid system. In brief 62.5 μCi of ³⁵S-CTP was added to reaction mixtures in a total volume of 20 μL consisting of 40 mmol/L Tris HCl, pH 7.5; 6 mmol/L MgCl₂; 2 mmol/L spermidine; 10 mmol/L NaCl; 10 mmol/L DTT; 1 U/μL RNase inhibitor; 0.5 mmol/L each of ATP, UTP, and GTP; 10 μmol/L unlabeled CTP; linearized template equivalent to 1 μg of whole plasmid; and 40 U of either T3 or T7 RNA polymerase. The reaction was allowed to proceed for 3 to 4 hours at 37°C. The DNA template was removed by addition of 1.5 U of RNase-free DNase for 30 minutes at 37°C. Proteins and free ribonucleotides were removed by phenol-chloroform–isoamyl alcohol extraction followed by ethanol precipitation. All probes were made to ≈350 bases long by controlled alkaline hydrolysis.

Whole bodies of mice were fixed by cardiac perfusion with 4% buffered paraformaldehyde after the animals had been anesthetized with ether. Hearts and lungs were removed, excised, and immersed overnight in the same fixative. Tissues were then embedded in paraffin, sectioned, and placed on Vectabond reagent (Vector Laboratories)–treated slides. After deparaffinization in xylene and hydration through a graded ethanol series, sections were digested with proteinase K (1 μg/mL) for 30 minutes at 37°C, washed in distilled water, and acetylated in freshly prepared acetic anhydride solution, pH 8.0, for 10 minutes. After one wash in 2× SSC, sections were dehydrated through a graded ethanol series. The hybridization mixture (600 mmol/L NaCl; 10 mmol/L Tris HCl, pH 8; 1 mmol/L EDTA; 50% formamide; 1× Denhardt’s solution; 10% dextran sulfate; 100 μmol/L DTT; 0.2% SDS; and 0.5 mg/mL yeast tRNA) containing 0.135 μg/mL of radiolabeled RNA probe was applied to each section and covered with a silanized coverslip. Hybridization was carried out for 16 hours at 45°C in light mineral oil. Excess probe was removed by washing in chloroform followed by washing for 1 hour in 4× SSC containing 1 mmol/L DTT. After dehydration, the slides were stringently washed at 60°C for 15 minutes in a buffer containing 300 mmol/L NaCl; 10 mmol/L Tris HCl, pH 8; 1 mmol/L EDTA; 50% formamide; and 100 μmol/L DTT. After a brief wash in 2× SSC, the slides were digested for 30 minutes at 37°C with 25 μg/mL RNase A in buffer containing 500 mmol/L NaCl; 10 mmol/L Tris HCl, pH 8.0; and 1 mmol/L EDTA. After a 30-minute wash at 37°C in the same buffer without the enzyme, sections were rinsed again in 4 L of 2× SSC and washed at high stringency in 0.1× SSC at 60°C, followed by dehydration through a graded ethanol series containing 300 mmol/L ammonium acetate and air drying before autoradiography. Autoradiography was performed by dipping the slides into NTB-2 autoradiography emulsion (Eastman Kodak) liquefied at 45°C and diluted 1:1 (vol/vol) in 600 mmol/L ammonium acetate. After they were dried, the slides were stored in light-proof boxes containing desiccant and exposed at 4°C for 6 to 10 days. The exposed slides were developed in Kodak D19 developer for 2.5 minutes, rinsed in 2% acetic acid for 30 seconds, and fixed in Rapid Fixer (Chugai Photo Chemical Co Ltd) for 5 minutes. After they were washed in tap water, the slides were counterstained with hematoxylin with or without eosin. Sections from untreated and IL-1β–treated mice were always processed in parallel with the same batches of probes and reagents.

Immunohistochemical Analysis

To obtain monoclonal antibodies against mouse E-selectin, cell membranes from a Chinese hamster ovary cell line (CHO K1) transfected stably with pEFneo expression vector (a kind gift from Dr A. Yoshimura) containing the mouse E-selectin cDNA were used for immunization of rats. Wistar rats (4 weeks old) purchased from Charles River Japan, Inc (Yokohama, Japan) were immunized once by foot pad subcutaneous injection with the Chinese hamster ovary cell membranes (prepared from 8×10⁶ cells) emulsified with an equal volume of Freund’s complete adjuvant (Chemicon Inc) and were repeatedly immunized (once a week for 1 month) with the membranes only. A hybridoma clone producing an IgM antibody that blocked adhesion of HL-60 promyelocytes to the Chinese hamster ovary cells expressing mouse E-selectin was the source of a monoclonal antibody named 67SG. Anti-mouse ICAM-1 monoclonal antibody (KAT-1) was purchased from Seikagaku Corp.

Five hours after administration of IL-1β (50 ng per mouse), the mice were killed; the hearts and the lungs were inflated with warm OCT compound and snap-frozen in LN₂. Sections of the frozen hearts were cut to be 4-μm thick by use of a cryostat and were mounted on glass slides. Sections were fixed in acetone for 5 minutes, air dried, and stained individually with anti–E-selectin (67SG) or anti–ICAM-1 (KAT-1) monoclonal antibody (5 mg/mL) for 45 minutes at room temperature. Staining for the presence of the indicator antibody was achieved with the Vectastain biotin/avidin peroxidase system (Vector Labs Inc). Sections were incubated with diaminobenzidine at 100 mg/mL for 5 minutes, counterstained with hematoxylin, and then permanently mounted with coverslips.

Distribution of Radioiodinated IL-1β

The mice were injected with radioiodinated, recombinant IL-1β via the tail vein with 5×10⁶ cpm (10 ng) of IL-1β and were killed 1 hour later. The radioactivity per milligram of wet tissue weight in the liver, kidney, heart, lung, brain, and skin was then determined in duplicate samples. Duplicate tissue samples of ≈60 to 300 μg were weighed and then minced before determination of radioactivity by gamma scintillation spectrometry.

Tissue-Specific Gene Expression of Adhesion Molecules in IL-1β–Treated Mice

C57BL/6 female mice were injected intravenously with IL-1β (50 ng per mouse) or vehicle (PBS). The animals were killed 2 hours later and samples of liver, kidney, heart, lung, brain, and skin were prepared. Total RNA isolated from each tissue was analyzed by Northern blot hybridization for specific mRNA content. Surprisingly, E-selectin mRNA expression was induced specifically in the heart. On the other hand, ICAM-1 mRNA expression was induced in almost all organs examined, although its strongest induction was seen in the heart and lung. Similar to ICAM-1 mRNA, VCAM-1 mRNA was also induced in various organs with the exception of the skin. Additionally, it is interesting to note that in the brain VCAM-1 mRNA showed stronger induction than did the two other adhesion molecules. α-Tubulin mRNA was expressed at almost equivalent levels in the different tissues examined, although slightly higher levels of expression were seen in the lung and brain. Thus, adhesion molecule gene expression differs, depending on the organs examined.

IL-1β–induced gene expression of adhesion molecules occurred rapidly (within 1 hour), transiently (nearly undetectable by 4 hours) in both the heart and lung, and with the same time course (Figure 2⇓). This pattern of expression was also seen in the kidney and liver (data not shown). As shown in Figure 1⇓, E-selectin mRNA expression was induced selectively in the heart, whereas ICAM-1 and VCAM-1 mRNAs were induced more abundantly in the lung and equivalently in both organs (also see Figure 2⇓).

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Figure 1.

Tissue distribution of gene expression of adhesion molecules in IL-1β–treated mice. C57BL/6 mice were injected with 50 ng per mouse of IL-1β in 0.1 mL of PBS or with PBS alone via the tail vein. Two hours later, the animals were killed and total RNA was prepared as described in “Methods.” Total RNA (20 μg) from each tissue was separated by denaturing agarose gel electrophoresis, transferred onto nylon membranes, and hybridized with³²P-labeled probes for murine E-selectin, ICAM-1, VCAM-1, or α-tubulin. Similar results were obtained in three experiments.

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Figure 2.

Time dependence of adhesion molecule mRNA expression in the murine heart and lung after systemic treatment with IL-1β. C57BL/6 mice were injected with 10 ng per mouse of IL-1β via the tail vein, and after the indicated times the animals were killed and total RNA was prepared from the heart and lung. Total RNA was analyzed by Northern blot hybridization with the indicated probes as described in the legend to Figure 1⇑. Similar results were obtained in two separate experiments.

Because the differential expression of adhesion molecules in the tissues was influenced by differential access of the injected stimulus to the tissue, the tissue distribution of IL-1β was determined 1 hour after intravenous injection of radioiodinated IL-1β by excising the different tissues and measuring their radioactivity on a per weight basis (the Table⇓). In contrast to increased gene expression of adhesion molecules in the heart, the accessibility of IL-1β to the heart was rather low, ie, approximately half the value of that in the liver, lung, or skin. Although IL-1β accumulated abundantly in the kidney, this accumulation might have become inactivated after its passage through metabolic processes, or it may have been stored in the kidney before excretion into the urine. On the other hand, IL-1β injected intravenously showed little access to the brain, probably owing to the blood-brain barrier.

To further examine the concentration dependence of IL-1β–driven mRNA expression in the heart and lung, a dose-response profile for intravenous IL-1β was obtained (Figure 3⇓). Treating animals with a series of increasing doses of IL-1β illustrated that the induction of E-selectin mRNA was dose dependent, with minimal response seen at 0.5 ng per mouse in the heart, although a slight induction of E-selectin mRNA in the lung was observed at 50 ng per mouse. Whereas ICAM-1 mRNA expression was augmented by IL-1β at 0.5 ng per mouse in both organs, the sensitivity of VCAM-1 mRNA expression in response to IL-1β was a little higher in the heart than in the lung.

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Figure 3.

Dose dependence of IL-1β–induced adhesion molecule mRNA expression in the murine heart and lung. C57BL/6 mice were injected with indicated amounts of IL-1β in 0.1 mL of PBS and killed 2 hours later. Total RNA was prepared from the heart and lung and analyzed as described in the legend to Figure 1⇑. Blots were hybridized with indicated radiolabeled probes. Similar results were obtained in three different experiments.

With respect to the observation that the accessibility of IL-1β to the heart was about half of that to the lung, the response of E-selectin mRNA induction in the heart was estimated to be ≈200-fold more sensitive than that in the lung. In the case of VCAM-1 mRNA induction, the heart was considered to be 20-fold more sensitive than the lung.

Greater Heart-Specific Induction of E-Selectin mRNA Expression After Treatment With IL-1β Than With TNF–α

To assess the stimulus specificities of adhesion molecule gene expression, we compared the effects of systemic treatment with IL-1β, TNF-α, or LPS in both the heart and lung. Mice were injected intravenously with IL-1β (10 ng per mouse), TNF-α (100 ng per mouse), or LPS (500 ng per mouse). Two hours later, total RNA was prepared from the heart and the lung and then examined for mRNA content (Figure 4⇓). Although treatment with TNF-α or LPS induced ICAM-1 and VCAM-1 gene expression in a similar manner to IL-1β treatment in both organs, induction of E-selectin mRNA expression by TNF-α or LPS was less marked than that by IL-1β. Thus, E-selectin expression in the heart differed, depending on the type of stimulus applied.

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Figure 4.

Effects of various stimuli on adhesion molecule mRNA expression in the murine heart and lung. C57BL/6 mice were injected with vehicle alone or with IL-1β (10 ng per mouse), TNF-α (100 ng per mouse), or LPS (500 ng per mouse) in 0.1 mL of PBS via the tail vein. Two hours later, the animals were killed; total RNA was prepared from the heart and lung and analyzed by Northern blot hybridization as described in the legend to Figure 1⇑. Similar results were obtained in three different experiments.

In Situ Hybridization Analysis of mRNA Expression of Adhesion Molecules

To determine the cell types responsible for gene expression of adhesion molecules, sections from the heart and lung were examined by in situ hybridization. Sections from the hearts and lungs of control and IL-1β- or TNF-α–treated animals were hybridized with³⁵S-radiolabeled sense or antisense cRNA probes for the adhesion molecules. Sense-strand probes hybridized poorly with sections from both untreated and treated mice (data not shown). Analysis of heart sections from IL-1β–treated animals by hybridization with the antisense E-selectin mRNA probe revealed markedly elevated signals (Figure 5C⇓ and 5D⇓), whereas sections from untreated animals showed weak signals (Figure 5A⇓ and 5B⇓), similar to those seen with sense-strand probes. Signals of E-selectin mRNA were detected not only in a diffuse, scattered pattern but also in a sporadically aggregated pattern. Close examination demonstrated that the grains with the latter pattern were localized over stromal cells with elongate nuclei and not over the cardiac muscle cells (Figure 5E⇓ and 5F⇓), suggesting that IL-1β treatment induced E-selectin mRNA expression not only in endothelial cells lining the capillary beds but also in other stromal cells. When TNF-α was substituted for IL-1β, localization of E-selectin mRNA expression was similar (data not shown). Analysis of lung sections from IL-1β–treated mice revealed no increased signal for E-selectin mRNA (data not shown). Diffuse signals of ICAM-1 mRNA in the hearts of IL-1β–treated mice were detected in a scattered pattern, probably due to the endothelial cells lining capillary vessels and not cardiac muscle cells (Figure 6⇓). Interestingly, endothelial cells lining the small arteries in the heart revealed induction of ICAM-1 mRNA expression, although they did not show detectable E-selectin mRNA. On the other hand, diffuse signals of ICAM-1 mRNA were also detected in the lungs of IL-1β–treated mice, including endothelial cells lining the blood vessels but not bronchial epithelial cells (Figure 7C⇓ and 7D⇓); close examination revealed that the cells composing the alveolar septa (other than endothelial cells) also expressed ICAM-1 mRNA (Figure 7E⇓ and 7F⇓). In contrast to E-selectin and ICAM-1 expression, VCAM-1 mRNA expression was observed mainly in endothelial cells lining the larger blood vessels in both the heart and lung in response to IL-1β treatment (Figure 8⇓). Endocardiac cells in both ventricles were also positive for VCAM-1 mRNA expression (data not shown).

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Figure 5.

In situ hybridization analysis of E-selectin mRNA expression in hearts from IL-1β–treated mice. C57BL/6 mice were untreated (A and B) or treated with IL-1β (50 ng per mouse) in 0.1 mL of PBS (C–F) and killed 1 hour later, followed by cardiac perfusion with 4% buffered paraformaldehyde. Tissue sections from the heart were hybridized with either sense (data not shown) or antisense cRNA probe for E-selectin radiolabeled with ³⁵S-CTP. After autoradiography, slides were lightly stained with hematoxylin and examined by bright-field (A, C, and E) or dark-field (B, D, and F) illumination. Magnification: ×62.5 (A–D), ×250 (E and F). White particles represent silver grains marking positive signals for E-selectin mRNA under dark-field illumination. Arrows indicate stromal cells with elongate nuclei on which silver grains were aggregated. Ar indicates small artery. Similar results were obtained in three experiments.

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Figure 6.

In situ hybridization analysis of ICAM-1 mRNA expression in hearts of IL-1β–treated mice. C57BL/6 mice were untreated (A and B) or treated with IL-1β (50 ng per mouse; C–F) and killed 1 hour later followed by cardiac perfusion with 4% buffered paraformaldehyde. Tissue sections from hearts were hybridized with either sense (data not shown) or antisense cRNA probe for ICAM-1 radiolabeled with ³⁵S-CTP. After autoradiography, the slides were lightly stained with hematoxylin and examined under light-field (A, C, and E) or dark-field (B, D, and F) illumination. Magnification: ×62.5 (A–D), ×125 (E and F). Ar indicates small artery. Similar results were obtained in three experiments.

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Figure 7.

In situ hybridization analysis of ICAM-1 mRNA expression in lungs from IL-1β–treated mice. C57BL/6 mice were untreated (A and B) and treated with IL-1β (C–F). One hour later, animals were killed, followed by cardiac perfusion with 4% paraformaldehyde. Tissue sections from lungs were hybridized with either sense (data not shown) or antisense cRNA probe for ICAM-1 radiolabeled with ³⁵S-CTP. After autoradiography, the slides were lightly stained with hematoxylin and eosin and examined under light-field (A, C, and E) or dark-field (B, D, and F) illumination. Magnification: ×62.5 (A–D), ×250 (E and F). Br indicates bronchus; BV, blood vessel; and Al, alveolus. Similar results were obtained in three experiments.

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Figure 8.

In situ hybridization analysis of VCAM-1 mRNA expression in hearts and lungs of IL-1β–treated mice. C57BL/6 mice were untreated (A and B) or treated with IL-1β (C–F) and killed 1 hour later, followed by cardiac perfusion with 4% paraformaldehyde. Tissue sections from hearts (A–D) and lungs (E and F) were hybridized with either sense (data not shown) or antisense cRNA probe for ICAM-1 radiolabeled with ³⁵S-CTP. After autoradiography, the slides were lightly stained with hematoxylin and examined under light-field (A, C, and E) or dark-field (B, D, and F) illumination. Magnification ×62.5. Ar indicates artery; BV, pulmonary blood vessel; and Br, bronchus. Similar results were obtained in three experiments.

To further demonstrate that the increases in message levels of adhesion molecules in response to proinflammatory stimuli were associated with increased expression of proteins, immunohistochemical studies for E-selectin and ICAM-1 were performed (Figure 9⇓). No staining was detected in tissue sections from IL-1β–treated mice without specific antibodies against the adhesion molecules (Figure 9A⇓). Analysis of heart sections from IL-1β–treated mice with anti–E-selectin antibody revealed that immunostaining was detected in a linear pattern parallel to the cardiac muscle cells (probably due to endothelial cells lining the capillary vessels) as well as sporadically over the cells with elongate nuclei (Figure 9C⇓ and 9D⇓), whereas heart sections from untreated mice showed few immunostained spots for E-selectin (Figure 9B⇓). Analysis of lung sections from the IL-1β–treated mice revealed low detection of E-selectin (data not shown). Immunostaining for ICAM-1 in the hearts of IL-1β–treated mice was detected in a linear pattern parallel to the cardiac muscle cells and also over the endothelial cells lining the small blood vessels (Figure 9E⇓). Immunostaining for ICAM-1 was also detected in the lungs of IL-1β–treated mice over all cells except bronchial epithelial cells (data not shown). These immunohistochemical studies demonstrate that the expression pattern of each adhesion molecule is identical with that of each message level.

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Figure 9.

Immunohistochemical analysis of E-selectin and ICAM-1 expression in hearts from IL-1β–treated mice. Five hours after administration of IL-1β (50 ng per mouse) or vehicle (PBS) alone, C57BL/6 mice were killed and their hearts removed. Sections of frozen hearts from untreated (B) or IL-1β–treated mice (A, C–E) were stained with anti-mouse E-selectin (B–D) or anti-mouse ICAM-1 (E) monoclonal antibody or without them (A), followed by staining of the indicator antibody with the biotin-avidin-peroxidase system. After incubation with diaminobenzidine, slides were counterstained with hematoxylin. Magnification, ×125 (A–C and E), ×250 (D). Arrows indicate stromal cells with elongate nuclei over which immunostaining of E-selectin was detected. V indicates small vein. Similar results were obtained in two different experiments.