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(Stroke. 1997;28:155-162.)
© 1997 American Heart Association, Inc.

Articles

Interleukin-1 Receptor and Receptor Antagonist Gene Expression After Focal Stroke in Rats

Xinkang Wang, PhD; Frank C. Barone, PhD; Nambi V. Aiyar, PhD Giora Z. Feuerstein, MD

the Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.

Correspondence to Xinkang Wang, PhD, Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd, PO Box 1539, UW2511, King of Prussia, PA 19406. E-mail xinkang_wang-1@sbphrd.com.

	Abstract

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Background and Purpose The expression of interleukin-1ß (IL-1ß) is upregulated after focal brain ischemia, and previous work has demonstrated its involvement in ischemic injury. The IL-1 receptor antagonist (IL-1ra), a natural competitive antagonist of IL-1 receptors (IL-1Rs), has been demonstrated to play a role in attenuating brain ischemic injury. To hypothesize the involvement of the IL-1 system in ischemic injury, we examined other IL-1 components, including IL-1ra, IL-1RI, and IL-1RII for their mRNA expression after focal stroke.

Methods Quantitative reverse transcription and polymerase chain reaction (RT-PCR) technique was used to examine the mRNA expression profile of IL-1ra and two IL-1R isoforms in a temporal fashion (n=4 for each time point) after permanent occlusion of the middle cerebral artery (MCAO) in spontaneously hypertensive rats. IL-1ra and IL-1R mRNA expression was confirmed by Northern blot analysis using poly(A) RNA isolated after 2 and 12 hours of MCAO.

Results Very low levels of IL-1ra mRNA were detected in sham-operated or nonischemic cortex. IL-1ra mRNA in ischemic cortex was greatly increased at 12 hours (16.5-fold increase over sham samples, P<.001) and remained elevated for up to 5 days (17.2-fold increase, P<.01) after MCAO. IL-1RI mRNA was relatively highly expressed in normal cortex and was further elevated late after ischemic injury (3.3-fold increase at day 5, P<.001). In contrast, the low basal expression of IL-1RII mRNA was remarkably elevated at 6 hours (5.3-fold increase, P<.05), reaching peak levels 12 hours (10.3-fold increase, P<.001) after MCAO.

Conclusions Differential expression of IL-1ß, IL-1ra, IL-1RI, and IL-1RII mRNAs after focal stroke may suggest a distinct role(s) for each component of the IL-1 system in ischemic injury. The data also stress the importance of evaluating all the components of a given cytokine system (eg, agonist, receptors, and natural antagonist) after focal stroke.

Key Words: cerebral ischemia, focal • cytokines • interleukins • rats

	Introduction

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Interleukin-1ß is a cytokine with multiple proinflammatory, procoagulant, and cell-growth modulatory actions.¹ IL-1ß can be synthesized by diverse cells in the central nervous system, including endothelium, microglia, astrocytes, and neurons.^{2 3} IL-1ß is present in the cerebral spinal fluid of normal brain and in disease states.⁴ Acute excess of IL-1ß in the brain has been suspected to result in proinflammatory events¹ that can promote neuropathological reactions. The increased expression of IL-1ß mRNA and/or protein has been demonstrated in brain after endotoxin administration,⁵ mechanical brain injury produced by intraparenchymal implantation of a microdialysis probe,⁶ and direct intraparenchymal administration of the neurotoxin kainate⁷ or other cytokines such as interferon-.⁸ Recently, the upregulation of IL-1ß mRNA has been observed in several animal models of brain injury, including trauma⁹ and ischemia.^{10 11 12 13 14 15}

The biological response to IL-1s is mediated by specific surface receptors. To date, two primary receptors for IL-1 have been identified that belong to the Ig superfamily.^{2 3} The extracellular segment of the two receptors contains three Ig-like domains and shares 28% amino acid sequence homology. The type I IL-1R (IL-1RI), an 80-kD glycoprotein, is prevalent in T cells, endothelial cells, smooth muscle cells, and fibroblasts, whereas the type II receptor (IL-1RII), a 68-kD protein, is found in B cells and macrophages.^{2 3} The cDNA encoding both receptors has been cloned from humans, mice, and rats.^{16 17 18 19} The major difference between the two receptors exists in their cytoplasmic domain, ie, the IL-1RI contains a larger cytoplasmic domain (213 amino acids) than IL-1RII (29 amino acids in humans and 35 in rats).² This difference allows IL-1RI but not IL-1RII to engage in intracellular signal transduction,^{3 20 21} while IL-1RII may act as a "sink" for IL-1ß.

IL-1ra, a 23- to 25-kD glycosylated protein, is a naturally occurring inhibitor of IL-1 activity that competes with IL-1 for occupancy of IL-1RI without inducing a signal of its own. IL-1ra has a higher binding affinity for IL-1RI than IL-1 and IL-1ß.³

A large number of studies indicate that both native and recombinant IL-1ra can block IL-1 activity in vitro and in vivo.³ For example, IL-1ra has been shown to block the activity of IL-1 in a number of animal disease models, such as septic shock, immune-complex–induced colitis, experimental diabetes, and reactivating arthritis (for reviews, see References 3 and 22). In addition, accumulating evidence demonstrates the protective effects of IL-1ra in brain injury. Thus, intracerebroventricular administration of recombinant IL-1ra produces a marked reduction in brain damage induced by focal stroke^{23 24} or fluid percussion injury in the rat.²⁵ This neuronal protective effect of IL-1ra in focal stroke was further supported by a recent study using an adenoviral vector that overexpressed IL-1ra in the brain.²⁶

These studies strongly suggest that IL-1ß plays a pivotal role in the pathogenesis of ischemic brain damage. In an effort to further characterize the involvement of the IL-1 system in focal ischemic injury, we investigated the temporal expression of IL-1ra, IL-1RI, and IL-1RII mRNA in rat focal stroke.

	Materials and Methods

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Focal Brain Ischemia
Cerebral focal ischemia or sham surgery was carried out in male SHR (16 to 18 weeks of age, weight 250 to 330 g; Taconic Farms, Germantown, NY) by permanent MCAO as described in detail previously.^{27 28} Briefly, the MCA was occluded and cut dorsal to the lateral olfactory tract at the level of the inferior cerebral vein using electrocoagulation (Force 2 Electrosurgical Generator, Valley Lab Inc). In sham-operated rats, the dura was opened over the MCA, but the artery was not occluded. Rats were later overdosed with pentobarbital, and forebrains were removed for cortical dissection at various times after MCAO. The ischemic frontoparietal cortex was dissected from the ipsilateral hemisphere. The contralateral cortex was dissected as the nonischemic control from the same rat.²⁷ The cortical samples were immediately frozen in liquid nitrogen and stored at -80°C.

RT-PCR
Total cellular RNA was prepared from cortical samples as previously described^{29 30} and subjected to quantitative RT-PCR analysis. Briefly, the cellular RNA (5 µg) isolated from the cortical samples at the indicated time points after MCAO was reverse transcribed with 200 U RNase H^- SuperScript II reverse transcriptase (Gibco BRL) for 60 minutes at 37°C, primed with 1 µg oligo(dT)_12-18 (Gibco BRL) at conditions recommended by the manufacturer. The RT products were extracted with phenol/chloroform, ethanol precipitated, resuspended in 200 µL Tris/EDTA (10 mmol/L Tris and 1 mmol/L EDTA, pH 7.5), and stored at -20°C.

The quantitative PCR was carried out as described in detail previously.³¹ A reference gene (rpL32 in this case) that has previously shown constant expression throughout the time course following MCAO^{30 31} was used as an internal control for the coamplification with the gene to be examined. PCR primers used for amplification of IL-1ra, IL-1RI, IL-1RII, and rpL32 were synthesized according to published sequences (Table). To define the optimal amplification conditions, we tested (1) a dose-dependent amplification of the RT-PCR using 25 to 800 ng RNA isolated from the rat ischemic cortex at 12 hours after MCAO, (2) effects of different cycles (15 to 40 cycles) of amplification for each primer pairs, and (3) the amount of ³²P-labeled primers for optimal detection of the coamplified genes. The representative data showing the coamplification of IL-1ra and rpL32 mRNA are illustrated in Fig 1. On the basis of these initial results, the linear portion of the amplification was determined for both the testing genes and the internal standard (ie, rpL32 in the present study). Therefore, the following conditions were chosen as standard for PCR reactions in a total 50 µL of reaction mixture: RT products from 0.1 µg RNA, 28 cycles for IL-1ra or 30 cycles for IL-1RI and IL-1RII. The reaction mixture contained 1x10⁶ cpm ³²P-labeled antisense primers for the examined genes and 5x10⁴ (for 28 cycles) or 4x10⁴ cpm (for 30 cycles) for rpL32, together with 100 ng each of nonradioactive sense and antisense primers (Table). The amplification was carried out using 2.5 U TaqAmpli polymerase (Perkin-Elmer Cetus) in a thermocycler (Perkin-Elmer Cetus) according to the conditions described previously^{31 34} : initial denaturation, 3 minutes at 94°C; initial annealing, 1 minute at 54°C; and initial extension, 3 minutes at 72°C. The subsequent cycles were denaturation, 15 seconds at 94°C; annealing, 20 seconds at 54°C; and extension, 1 minute at 72°C. The PCR product (10 µL) was electrophoresed through a 6% polyacrylamide gel. The gel was dried and subjected to autoradiography at room temperature. The signal intensity was quantified using PhosphorImager (Molecular Dynamics) analysis, and the relative mRNA levels were determined by calculating the ratio of IL-1ra, IL-1RI, or IL-1RII to rpL32 in each coamplified sample.

View this table: [in this window] [in a new window]   Table 1. Oligonucleotide Primers of IL-1ra, IL-1RI, IL-1RII, and rpL32 Used for PCR*

View larger version (17K): [in this window] [in a new window]   Figure 1. Quantification of coamplified IL-1ra and rpL32 mRNA in ischemic cortex 12 hours after MCAO. RT-PCR was performed using the standard conditions as described in detail in "Materials and Methods" except that different amounts of RT products (0.025 to 0.8 µg per reaction) (A), numbers of cycles (15 to 40) (B), or labeled primers (C) were applied for the amplification. PCR products were resolved by electrophoresis, and the band intensity was measured using PhosphorImager analysis. The relative intensity was illustrated as the percentage of signal for either IL-1ra or rpL32 over the sum of each condition.

Northern Blot Analysis
To confirm the quantitative RT-PCR data, we applied Northern blot analysis using poly(A) RNA isolated from ischemic and nonischemic cortex of 50 rats at either 2 or 12 hours after MCAO. Poly(A) RNA (10 µg per lane) was electrophoresed through formaldehyde agarose gel and transferred to a GeneScreen Plus membrane (Du Pont-New England Nuclear). For Northern analysis, the cDNA fragments for IL-1ra, IL-1RI, IL-1RII, and rpL32 were gel purified after PCR amplification (as described above) and were uniformly labeled with [-³²P]dATP (3000 Ci/mmol, Amersham Corp) using a random-priming DNA labeling kit (Boehringer Mannheim). Hybridization and washing were performed as described in detail previously.^{30 34} The rpL32 cDNA probe was added together with the testing gene as a loading control.

Statistical Analysis
Statistical evaluation was performed using four complete sets of cortical samples from each time point using one-way ANOVA followed by Fisher's protected t test. The results are expressed as mean±SE. Significance was accepted for P<.05 by comparing the relative mRNA levels in the ischemic cortex with the sham-operated cortex.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Fig 2A illustrates a representative autoradiograph of an RT-PCR experiment to detect IL-1ra mRNA expression in the focal ischemic and nonischemic cortex and in sham-operated cortical samples. The quantitative data for IL-1ra mRNA (n=4), after normalizing to the internal control rpL32, are summarized graphically in Fig 2B. Sham-operated samples were taken at 3 and 12 hours and 5 days, and no difference was observed for IL-1ra (or IL-RI and IL-RII) mRNA expression. Therefore, only the sham samples of 12 hours are shown in the present study. As shown in Fig 2, only a low level of IL-1ra mRNA was detected in the sham-operated animals or in the contralateral (nonischemic) cortex, as well as at the early time points in the ipsilateral (ischemic) cortical samples. The level of IL-1ra mRNA was markedly increased in the ischemic cortex at 6 hours (6.0-fold increase compared with the sham samples, n=4), then reached a significantly elevated level from 12 hours (16.5-fold increase, P<.001) to 5 days (17.2-fold increase, P<.001) after MCAO (Fig 2).

View larger version (32K): [in this window] [in a new window]   Figure 2. Temporal expression of IL-1ra mRNA in ischemic cortex after MCAO using RT-PCR analysis. A, Representative autoradiograph showing the IL-1ra mRNA expression after MCAO. PCR was carried out according to the standard conditions as described in "Materials and Methods." The PCR products (10 µL per lane) were resolved by electrophoresis in a 6% polyacrylamide gel, dried, and autoradiographed. Ipsilateral and contralateral cortex samples (denoted by +) from individual rats after sham surgery (S; 12 hours after surgery) or following 1, 3, 6, 12, and 24 hours or 2 and 5 days of MCAO are depicted. The amplified cDNA fragments for IL-1ra and rpL32 had expected sizes of 525 and 412 bases, respectively, as resolved by molecular weight markers (not shown). B, Quantitative data showing the time course of the relative IL-1ra mRNA levels after MCAO. The coamplified DNA bands of IL-1ra and rpL32 were quantified by PhosphorImager analysis. The ratio of IL-1ra/rpL32 in each coamplified sample was calculated, and the relative levels of IL-1ra mRNA (as determined by their ratios) were illustrated. Data are presented as the mean±SE of four separate experiments in SHR (n=4) for each time point. **P<.01 and ***P<.001, compared with sham-operated (12 hours) cortex samples.

The temporal mRNA expression for IL-1RI and IL-1RII was investigated using the same samples as applied for IL-1ra. The representative autoradiograph of IL-1RI mRNA after focal brain ischemia is shown in Fig 3A, and corresponding quantitative data are illustrated in Fig 3B. Overall, there is a relatively high basal expression of IL-1RI mRNA in the cortical samples. However, a trend for the ischemia-induced IL-1RI mRNA expression was observed at 2 days after MCAO (1.7-fold increase compared with sham). A significant upregulation of IL-1RI mRNA was observed at 5 days after MCAO (3.3-fold increase, P<.001, n=4). In contrast, the ischemia-induced expression of IL-1RII mRNA was robust (Fig 4). IL-1RII mRNA expression was rapidly induced by MCAO, with increases of 2.3-fold at 3 hours and 5.3-fold at 6 hours (P<.05), reaching a peak level at 12 hours (10.3-fold increase, P<.001) and maintaining a significantly elevated level up to 2 days (8.6-fold increase, P<.01) after MCAO (Fig 4).

View larger version (31K): [in this window] [in a new window]   Figure 3. Temporal expression of IL-1RI mRNA after focal brain ischemia using RT-PCR analysis. Samples are illustrated as described in Fig 2, except that IL-1RI was coamplified with rpL32, and 30 cycles were applied as described in "Materials and Methods." The amplified IL-1RI cDNA had the expected size of 641 bases. The data are based on four independent experiments (n=4 for each time point) of RT-PCR. ***P<.001, compared with sham-operated (S; 12 hours after surgery) samples.

View larger version (33K): [in this window] [in a new window]   Figure 4. Temporal expression of IL-1RII mRNA in rat cortex after MCAO using RT-PCR analysis. RT-PCR was carried out as described in "Materials and Methods" and as illustrated in Fig 2. IL-1RII was coamplified with rpL32 for 30 cycles. The PCR product of IL-1RII had the expected size of 530 bases. The data are based on four independent RT-PCR experiments (n=4 for each time point) as described in Fig 2. *P<.05, **P<.01, and ***P<.001, compared with sham-operated (S; 12 hours) samples.

The expression of IL-1ra, IL-1RI, and IL-1RII mRNA in the ischemic and nonischemic cortex was confirmed by Northern analysis using selected time points, ie, 2 and 12 hours after permanent MCAO (Fig 5). The Northern hybridization data were in agreement with the data generated by quantitative RT-PCR. As shown in Fig 5, only a trace level of IL-1ra and IL-1RII mRNA was observed in the nonischemic cortex. Ischemia induced a low level of expression of these two messengers at 2 hours but a marked induction at 12 hours after MCAO. As predicted, the level of IL-1RI mRNA showed little change at these early time points.

View larger version (33K): [in this window] [in a new window]   Figure 5. Northern analysis of IL-1ra (A), IL-1RI (B), and IL-1RII (C) mRNA expression in rat cortex after MCAO. Poly(A) RNA (10 µg per lane) was resolved by electrophoresis, transferred to a nylon membrane, and hybridized to the cDNA probes as indicated. The positions of 28S and 18S ribosomal RNA were marked on the right. The upper arrow in each panel indicates IL-1ra (A), IL-1RI (B), and IL-1RII (C) mRNA, respectively, and the lower arrows refer to the rpL32 mRNA. Lane 1, 2 hours ischemic (ipsilateral) cortex; lane 2, 2 hours nonischemic (contralateral) cortex; lane 3, 12 hours ischemic cortex; and lane 4, 12 hours nonischemic cortex after MCAO.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Increased IL-1ß mRNA expression after brain ischemia has been demonstrated repeatedly.^{10 11 12 13 14} The exacerbation of brain injury due to exogenous IL-1ß administered into the brain has also been observed.¹⁵ IL-1ß, together with TNF-,^{11 35} is thought to play a key role in the inflammatory response after ischemic brain injury.³⁶ The elevated expression of these inflammatory cytokines, as well as adhesion molecules (eg, intracellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and P-selectin^{30 31 37} ) and chemokines (eg, KC and monocyte chemoattractant protein-1^{38 39} ), after focal brain ischemia may contribute to the recruitment of leukocyte into the ischemic brain and exacerbate ischemic injury.^{40 41 42}

The presence of IL-1ra in the normal brain and the upregulation of IL-1ra mRNA in the ischemic cortex suggest that IL-1ra can serve as a defense system to attenuate inflammatory reactions elicited by brain injury. The significant neuronal protection of IL-1ra has been demonstrated in animal models of focal brain ischemia,^{23 24 26} traumatic brain injury,²⁵ or in perinatal hypoxic brain damage.⁴³ It is interesting to observe that the temporal induction profile of IL-1ra after MCAO virtually parallels that of IL-1ß as demonstrated previously,¹¹ except that IL-1ra mRNA exhibited a more prolonged elevation up to 5 days after MCAO (Fig 6). It is possible that the induced expression of IL-1ra may interfere with IL-1ß–mediated effects after ischemic injury.

View larger version (17K): [in this window] [in a new window]   Figure 6. Temporal relationship of IL-1ß, IL-1ra, IL-1RI, and IL-1RII regulation in brain ischemia. The data were generated from the same rat model of MCAO. The temporal expression profiles of IL-1ra, IL-1RI, and IL-1RII are illustrated based on the present study using RT-PCR analysis. The temporal expression of IL-1ß mRNA was determined by Northern analysis,11 and the profile of leukocytes in the ischemic cortex was measured by myeloperoxidase activity assay and histological verification27 according to previous reports. PMN indicates polymorphonuclear; MONO/MØ, monocytes/macrophages.

The mediators responsible for IL-1ra induction after focal stroke are not known. However, previous studies indicate that some cytokines such as IL-1, TNF, IL-6, and TGF-ß are inducers of IL-1ra.³ The mRNA expression profiles of IL-1ß, TNF-, and IL-6 are remarkably parallel^{11 35 44} (Fig 6), whereas that of TGFß is slightly delayed, not significantly upregulated until 2 days after focal ischemia.⁴⁵ Interestingly, the temporal expression of these genes revealed a concomitant upregulation with IL-1ra after focal brain ischemia.

The cellular sources of IL-1ra expression after focal stroke have not been determined in the present studies. In normal brain, IL-1ra mRNA was not detected in the cortex, although its expression has been observed in the paraventricular nucleus of the hypothalamus, hippocampus, and cerebellum by in situ hybridization.⁴⁶ It is possible that the ischemia-induced expression of IL-1ra mRNA could be from monocytes/macrophages, endothelial cells, fibroblasts, neurons, and glial cells, as observed previously under normal conditions,^{2 3 22} or from the same cellular sources of IL-1ß expression because of their close temporal and perhaps functional correlation.

It is interesting that IL-1RI and IL-1RII mRNA expression is differentially regulated after focal brain ischemia. This difference might reflect the overall spatial and temporal differences in the expression of these two genes. For example, IL-1RI has been found mainly in T cells, endothelial cells, and fibroblasts, whereas IL-1RII has previously been localized to B cells, neutrophils, and macrophages.³ In addition, IL-1RI exhibits a relatively high basal expression in normal brain but IL-1RII does not. The expression of IL-1RI mRNA has been previously localized in cerebellar and hippocampal neurons, in choroid plexus, in the endothelium of postcapillary venules, and in glial cells surrounding arterioles throughout the brain.^{47 48} The high basal cortical expression of IL-1RI mRNA in the present work is in agreement with these previous studies. In addition, IL-1 binding activity in the brain has also been detected.^{49 50 51} Differences in IL-1R expression after focal stroke may reflect their distinct roles in ischemic injury. For example, IL-1RI but not IL-1RII stimulates IL-1–mediated signal transduction. Also, IL-1RI has the highest binding affinity for IL-1ra, whereas IL-1RII more readily binds IL-1ß.³ The remarkable parallel temporal expression of IL-1RI mRNA and leukocyte infiltration after MCAO (Fig 6) suggests that the upregulation of this signal-transducing receptor may be responsible for the IL-1ß–mediated leukocyte recruitment after ischemic insult. It is also interesting that the expression pattern for IL-1RII mRNA is remarkably parallel to that of IL-1ß mRNA after MCAO (Fig 6). Because IL-1RII (soluble or membrane-bound form) binds IL-1ß with a higher affinity but without transducing a signal, the upregulation of IL-1RII after focal stroke might provide an action similar to that of IL-1ra, ie, it could provide a natural compensatory mechanism to counter the activity of IL-1ß.

In addition to IL-1ß, IL-1ra, and IL-1Rs, other members of the IL-1 system have been identified and play important roles in IL-1–mediated actions, including IL-1 and IL-1R accessory protein. Our studies (X.W., F.C.B., N.V.A., G.Z.F., unpublished data, 1997) indicate that IL-1 gene expression, unlike that of IL-1ß, is not affected by ischemic stroke. Instead, IL-1 mRNA expression exhibits a constitutive expression pattern in both ischemic and nonischemic cortex after focal stroke. IL-1R accessory protein, which is similar to the IL-1Rs containing three Ig-like domains in the extracellular segment,³ is thought to play a functional role in IL-1 signal transduction: IL-1 binds IL-1RI with a low affinity, but this binding affinity is markedly increased when IL-1R accessory protein forms a complex with IL-1/IL-1RI.³ Unfortunately, the rat homologue of IL-1R accessory protein has not been cloned; therefore, no tool is available to evaluate this gene regulation after rat MCAO.

It also should be pointed out that the RT-PCR detection method used in the present report is very sensitive but only semiquantitative, since the amplification rates for the IL-1 system and the reference gene (ie, rpL32) are not identical (Fig 1). To provide a relatively accurate quantitation, it is critical to determine the linear range of the amplification for both the detecting gene and the reference gene and then coamplify them under these particular conditions.^{31 52 53} Comparatively, other techniques such as competitive PCR^{54 55} appear to be more attractive, but they are labor-intensive and unable to correct the differences between each sample started from RT. Thus, to evaluate the quantitative data generated by PCR in this report, we applied Northern blot analysis as shown in Fig 5, which is in agreement with the RT-PCR data.

In conclusion, the present data demonstrated a kinetic induction profile for IL-1ra, IL-1RI, and IL-1RII mRNA in the ischemic cortex and suggested the active involvement of the IL-1 system in focal stroke. The induced expression of IL-1ß along with IL-1RI, especially the parallel pattern of IL-1RI upregulation with leukocyte infiltration (Fig 6), strongly suggests a role of IL-1ß in facilitating leukocyte infiltration/accumulation into the ischemic tissue. Meanwhile, the upregulation of the endogenous IL-1ra and IL-1RII after focal stroke may attenuate this effect. However, it should be emphasized that the significance of the mRNA expression for the IL-1 system described in the present study must be interpreted with caution, since no evidence has been provided for an increase in the corresponding protein levels. Moreover, IL-1ß is not the only cytokine exhibiting increased production and contributing to ischemic injury. Rather, IL-1ß, together with other cytokines (such as TNF-^{12 35} ), plays a role in leukocyte recruitment after focal stroke. Nevertheless, the present study suggests the importance of studying all the components of a given cytokine system (eg, cytokine, endogenous antagonist, and receptors) in the process of developing ischemic brain injury.

	Selected Abbreviations and Acronyms

 

IL-1ß = interleukin-1ß IL-1R = interleukin-1 receptor IL-1ra = interleukin-1 receptor antagonist MCA(O) = middle cerebral artery (occlusion) PCR = polymerase chain reaction RT = reverse transcription SHR = spontaneously hypertensive rat(s) TGF = transforming growth factor TNF = tumor necrosis factor

Received May 23, 1996; revision received August 30, 1996; accepted September 26, 1996.

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Editorial Comment

Gregory J. del Zoppo, MD, Guest Editor

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, Calif

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Cytokine synthesis and release are important events in the development of focal cerebral ischemia. Descriptions of TNF-,^{1R 2R} IL-1ß,^{2R 3R} IL-6,^4R and monocyte chemoattractant protein-1^5R messenger RNA expression after MCAO in the rat have recently appeared. The similarity in time course of upregulation has suggested a global stimulation of the inflammatory system. Sources of the active proteins have also been sought. Liu and colleagues^1R have described the presence of TNF- antigen on neuron fibers and other cells in the same species. Production of cytokine and chemokine mRNA coincident with or preceding polymorphonuclear (PMN) leukocyte and monocyte invasion in experimental focal ischemia has suggested a direct causal relation. TNF- and IL-1 are also known to stimulate P-selectin, intercellular adhesion molecule-1 and E-selectin expression in microvascular endothelial cells. The appearance of all three adhesion receptors in a timely sequence, consistent with PMN leukocyte adhesion and transmigration, has been described in focal cerebral ischemia.^{6 7} One attraction in defining the participation of cytokines in these processes is that blockade of steps upstream of PMN leukocyte activation, adhesion, and invasion may serve to reduce ischemic injury.

In this respect, the fate of IL-1 is of particular interest. Given the role of IL-1ß in cerebral inflammatory and procoagulant events,⁸ it is likely that synthesis of the cytokine should play a role in the inflammatory lesion after focal cerebral ischemia. Wong et al⁹ have described the synthesis of IL-1ß–converting enzyme (ICE), which converts IL-1ß into bioactive IL-1ß, in blood vessels throughout the brain. IL-1, IL-1ß, IL-lra, and the IL-1 type receptor are also synthesized in brain vasculature. The agonist, antagonist, and receptors appear to be intimately associated with the cerebral microvasculature and may act in an autocrine fashion to stimulate adhesion receptor synthesis under specific conditions. Intracerebroventricular infusion of purified IL-1 produces a significant reduction in the region of cerebral injury at 24 hours after MCAO in the rat,^10R indicating the relevance of the IL-1 agonist-antagonist axis to postischemic injury in the brain. The further work by Wang et al reported here extends our view of the potential interplay of the components of this axis through the response of their mRNA transcription to focal ischemia. They report that in response to focal ischemia, IL-1RII mRNA reached maximal expression by 12 hours after MCAO, in keeping with earlier reports of IL-1 mRNA elevation. The receptor antagonist IL-lra followed a similar profile with maximal induction by 12 hours and sustained expression. IL-1RI displayed a more gradual increase in expression. These findings imply that transcription of mRNAs specific for the receptor and the receptor antagonist have unique time-course profiles consistent with IL-1 expression. These data suggest that the IL-1 axis, and particularly IL-1ß, are coordinately expressed in response to focal ischemia.

Two caveats should be addressed to this work. The first is that transcription of component mRNAs does not necessarily reflect the levels of active cytokines or the presence of their receptors or their antagonists. The conversion of IL-1ß to its active form is dependent on the presence of active ICE. Therefore, the local levels of the proteins in relation to their transcripts should illuminate the activity of this part of the cytokine picture. The second is that RT-PCR techniques are elegant approaches to exploring transcripts present at low levels, but they remain semiquantitative, as pointed out by the authors. This is of particular interest because the coamplification of the desired transcript and a housekeeping gene depend on conditions that may vary for each transcript of interest (Fig 1). While RT-PCR may indicate a relative transcript level, it cannot identify the in situ location of the transcript.

Given the increasing interest in the fate of ICE in cerebral ischemia and the apparent complexity of the responses of the IL-1ß axis components as demonstrated here, knowing the location of cells that are responsible for these transcripts and their responses to focal ischemia will add measurably to our knowledge of how ischemia triggers the subsequent inflammatory events that lead to cerebral infarction.

	Selected Abbreviations and Acronyms

 

IL-1ß = interleukin-1ß IL-1R = interleukin-1 receptor IL-1ra = interleukin-1 receptor antagonist MCA(O) = middle cerebral artery (occlusion) PCR = polymerase chain reaction RT = reverse transcription SHR = spontaneously hypertensive rat(s) TGF = transforming growth factor TNF = tumor necrosis factor

S and A indicate sense and antisense oligonucleotides, respectively.

*Base pair positions are those given in the published cDNA sequences for rat IL-1ra,³² IL-1RI,¹⁸ IL-1RII,¹⁹ and rpL32.³³

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1R. Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, Feuerstein GZ. Tumor necrosis factor- expression in ischemic neurons. Stroke.. 1994;25:1481-1488.

2R. Wang X, Yue T-L, Barone FC, White RF, Gagnon RC, Feuerstein GZ. Concomitant cortical expression of TNF- and IL-1ß mRNA following transient focal ischemia. Mol Chem Neuropathol.. 1994;23:103-114.

3R. Liu T, McDonnell PC, Young PR, White RF, Siren AL, Hallenbeck JM, Barone FC, Feuerstein GZ. Interleukin-lß, mRNA expression in ischemic rat cortex. Stroke.. 1993;24:1746-1751.

4R. Wang XK, Yue T, Young PR, Barone FC, Feuerstein GZ. Expression of interleukin-6, c-fos and zif268 mRNAs in rat ischemic cortex. J Cereb Blood Flow Metab.. 1995;15:166-171.

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6R. Okada Y, Copeland BR, Mori E, Tung M-M, Thomas WS, del Zoppo GJ. P-selectin and intercellular adhesion molecule-1 expression after focal brain ischemia and reperfusion. Stroke.. 1994;25:202-211.

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9R. Wong ML, Bongiorno PB, Gold PW, Licinio J. Localization of interleukin-1 beta converting enzyme mRNA in rat brain vasculature: evidence that the genes encoding the interleukin-1 system are constitutively expressed in brain blood vessels: pathophysiological implications. Neuroimmunomodulation.. 1995;2:141-148.[Medline] [Order article via Infotrieve]

10R. Loddick SA, Rothwell NJ. Neuroprotective effects of human recombinant interleukin-1 receptor antagonist in focal cerebral ischaemia in the rat. J Cereb Blood Flow Metab. 1996. In press.

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