Enhancement of immunoreactivity for NF-κB in human cerebral infarctions

BRAIN RESEARCH BrainResearch739(1996)343-349

Short communication

Enhancement of immunoreactivity for NF-KB in human cerebral infarctions Kazuhiro Terai, Akinori Matsuo, Edith G. McGeer, Patrick L. McGeer * Kinsmen Laboratory of Neurological Research, The UniuersiQ of British Columbia, 2255 Wesbrook Mall, Vancouuer BC, V6T 123, Canada

Accepted27 August1996

Abstract The distribution of the nuclear factor-KB (NF-KB) was investigated immunohistochemically in recently infarcted areas of postmortem human brain. Previously we reported that immunoreactivity for NF-KB was enhanced in neurons of Alzheimer disease brain in comparison with control cases. In the present study, a similar enhancement of immunoreactivity was observed in glial cells of infarcted areas, but not in the unaffected surround. Prominent staining for NF-KB was seen in some astmcytes, particularly in the penumbra or border zone between ischemic and non-ischemic areas. In some cases, positively stained macrophages were also observed in affected ureas. Capillary staining for NF-KB was weak and did not differ significantly between affected and unaffected areas. These results suggest that enhanced expression of astrocytic NF-KB occurs in cerebral infarcted areas. Keywords; Penumbra;NF-KB; Microphage;Microglia;Astrocyte

Nuclear factor-KB (NF-KB) is a transcription factor involved in the positive regulation of cytokines [17,22], MHC class 1 and class 2 glycoproteins [3,4], and adhesion molecules [38]. It is induced by exposure to stimulants such as lipopolysaccharides [32] and inflammatory cytokines [27], and is currently regarded as a prominent regulator of inflammatory, immune and acute phase reactions. NF-KB is a heterodimer composed of p50 and p65 subunits. Its activation and regulation are tightly controlled by IKB, the endogenous inhibitor of NF-KB. The p65 subunit has a nuclear translocation signal sequence which is normally masked in the cytosol by IKB binding [11,29]. Release of NF-KB is initiated when specific kinases, activated by cell surface receptors, phosphorylate IKB. Proteolysis of IKB then occurs, releasing a heterodimer consisting of the p65 and p50 subunits of NF-KB, permitting translocation to the nucleus [5]. There the signal sequence of p65 binds to a specific DNA sequence called the B motif [33], whicb is the gene expression enhancer. More details of the NF-KB system can be found in review articles [5,21]. Cerebral infarction occurs when a vascular accident dramatically reduces blood flow to a particular area of

“ Corresponding author, Fax: + 1 (604) 822-7086; E-mail: mcgeerpl@unixg. ubc.ca

brain. As a result, the area becomes ischemic, leading to edema and infiltration of leukocytes. Damage to neurons occurs with subsequent astrogliosis. Although histopathological changes following cerebral infarction have been investigated extensively [8,16], the molecular mechanisms underlying repair are still not fully understood. Recent reports suggest that various cytokines [6,39] and adhesion molecules [14,26] play significant roles in the inflammatory and repair processes following upon ischemic brain changes (see [12] for review). Since NF-KB activity may be involved in some of these activities, we investigated whether the expression of NF-KB might be altered in infarcted areas of human brain. The six autopsied human brains employed in this study were obtained within 8–46 h of death (Table 1). Infarcted areas were identified in each of these cases: four cases with Alzheimer’s disease and two cases without dementia. The estimated time of occurrence of the cerebrovascular accident was determined from histological findings using the criteria established by Chuaqui et al. [8]. They identified four stages following an ischemic insult which were identifiable according to distinct histological features: the first stage, from day 1 through day 4, was characterized by a predominance of eosinophilic neurons and necrotic oligodendrocytes; the second stage, from day 5 through day 7, differed from the first by the appearance of macrophages and newly formed blood vessels; the third stage, from day 8 through day 14, showed neuronal ghosts, macrophages,

0006-8993/96/$15.00Copyright0 1996ElsevierScienceB.V. All rights reserved. 1073-6

P[[ S0006-8993(96)0

76 69 86 86

82 84

Male Male Male Female

Male Female

Fourth Fourth Fourth Third Third Third

MTG MTG, PCG, OCC ANG HIP CP, HIP HIP, OCC

Alzheimer Oesophagus cancer Alzheimer Metastatic thyroid cancer Alzheimer Alzheimer

10 8 14 20

10 46

Infarction stage

Damaged areas

Other disorders

Postmortem delay (hours)

++ +/+ + +/–

+ + +/– + + +/– + ++/– +++/–

Astrocytes

+/– ++/–

-/-/-/+/–

Macrophages

+/+ +/+

+/+ +/+ +/+ +/+

Vascular walls

Immunoreactivity for NF-KB (affected/unaffected areas in infarction)

The infarction stages were identified according to the criteria established by Chuaqui et al. [8]; the third stage (from day 8 to day 14), the fourth stage (from day 15 to day 27). —, not detected; +, low; + +, moderate; + + +, high intensity of immunostaining for NF-KB.Valuationis given for astrocytes, microphage-lilce cells (macrophages) and vascular walls in areas affected and unaffected by cerebral infarction. MTG,middletemporalgyms:PCG,precentralgyms:OCC,occipitalcortex;ANG, angular cortex; HIP, hippocampus; CP, caudate putamen.

Age

sex

Clinical and pathological features

Table 1 Summaryof clinicalfeaturesand NF-KBimmunohistochemistry in the cerebralinfarctioncases used in this study

*

K. Terai et al. /Brain Research 739 (19%) 343–349

astrocytic proliferation, gemistocytes, and art absence of neutrophils; and the fourth stage, from day 15 through day 27, demonstrated no eosinophilic neurons, and, in the central portion of the infarct, an absence of myelin and necrotic oligodendrocytes. The infarctions identified in our cases matched the third and fourth stages (i.e. from 8 to 27 days) (Table 1). The diagnosis of cerebral infarction was confh-rned in every case by the demonstration of severe neuronal damage, astrogliosis and penetration of leukocytes or macrophages as determined by HE and Cresyl violet staining. The border zone of the unaffected and the ischemic region, the penumbra, was also identified. Small brain blocks of approximately 5 mm thickness were dissected and immediately fixed in 470 paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 for 2 days. They were then transferred to a maintenance solution of 1570 sucrose in 0.1 M phosphate buffer, pH 7.4. Sections were cut on a freezing microtome at 30 p,m thickness, collected in the maintenance solution, and stored until stained. For immunohistochemical localization of the NF-KB, two kinds of well-characterized commercial rabbit polyclonal antibodies were used. One was for the NF-KB p65 subunit (diluted 300), and the antibody was raised against a synthesized peptide corresponding to amino acid residues

345

531–550 of the p65 subunit of human NF-KB (sc-372; Santa Cruz Biotechnology). The synthesized antigen peptide (control peptide; Santa Cruz Biotechnology) was used for immunoabsorption testing. Another antibody for NF-KB p50 (diluted 3000) was raised against a synthesized peptide corresponding to amino acids 350–363 mapping within the nuclear localization sequence region in the p50 subunit of human NF-KB (sc-1 14; Santa Cruz Biotechnology). To identify astrocytes, a rabbit antiserum against glial fibrillary acidic protein (GFAP) (Dakopatts, 1:10000) was utilized and, for macrophages, a mouse monoclinal antibody against HLA-DR (HB 104; American Type Culture Collection, 1:1000). These antibodies have been routinely used for astrocyte or microphage detection [20,34,37,40]. Immunohistochemistry was performed on free-floating sections. Prior to staining, sections were treated for 30 min with 0.270 hydrogen peroxide to eliminate endogenous peroxidase activity, For all subsequent procedures except the final DAB reaction, the buffer solution used was 0.1 M phosphate buffered saline containing 0.3% Triton X-100, pH 7.4 (PBS-T). Following preincubation for 1 h at room temperature with a blocking solution containing 5% normal serum of the secondary antibody host in PBS-T, incubation with the primary antibody was carried out for 3

Fig. 1. Comparisonof the immunohistochemical distributionpatternof the p65(A) andp50(B) subunitsof NF-KBin nearby sections of a cerebral infarct. Arrowheads indicate the same blood vessel. C and D: immunoabsorption test for the p65 subunit antibody. Immunohistochemical staining shows that the (C),butthestaining disappears whentheantibody is w=bsorbedwithitsankenic antibody strongly identities positive structures including askocytes peptide (20 ~g/ml) (D). Scale bar= 200 pm.

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K. Terai et al. /Brain Research 739 (1996) 343-349

days at 4“C. Sections were then treated for 2 h at room temperature with the appropriate biotinylated secondary antibody diluted 1:1000, followed by incubation for 1 h at room temperature in avidin-biotinylated HRP complex diluted 1:4OOO (ABC Elite system, Vector Lab., Burlingame, CA). Peroxidase labelling was detected by incubating with 50 mM Tris-HCl, pH 7.6 containing O.01% 3,3’-diaminobenzidine (DAB, Sigma), 0.6% nickel ammonium sulfate, 0.05 M imidazole and 0.00015% hydrogen peroxide [18]. A purple reaction product appeared after about 15–20 rein, at which time the reaction was terminated by transferring the sections to PBS-T. Sections were subsequently mounted on glass slides, dehydrated, and coverslipped with Entellan (Merck), with or without Neutral red counterstaining. Immunostaining for GFAP or HLA-DR was performed on nearby sections to those used for NF-KB staining. An immunoabsorption immunohistochemical test was performed against the p65 subunit. The antigen peptide was diluted in PBS-T at several concentrations ranging from 0.2 to 20 pg/ml. The solutions were added to the

NF-KB antibody (1:300). The mixtures were incubated for 12 h at 4°C and used for standard immunohistochemical staining. The immunohistochemical distribution patterns for the p65 and p50 subunits of NF-KB in neighboring infarcted brain sections were highly similar, as would be expected of a heterodimer consisting of these subunits (cf. Fig. IA,B). In immunoabsorption immunohistochemical testing, increasing the concentration of the p65 peptide antigen decreased the intensity of staining. At 20 pg/ml peptide, there was complete disappearance of any positive staining for the p65 subunit of NF-KB (cf. Fig. IC,D). An absence of staining also occurred when either NF-KB primary antibody was omitted. Decreasing the concentration of the primary antibodies significantly decreased the intensity of tissue imrnunostaining (data not shown). The specificity of the p65 antibody by Western blots of human brain homogenates was established in a previously published report [36]. Strong NF-KB staining of astrocytes was observed in all

Fig. 2. Morphological features and localization of astrocytes stained for NF-KB in representative infarcted sites. Positively stained astrocytes are always of reactive morphology (P65 subunit, A–D; P50 subunit, E and F). They are preferentially located in the penumbra, which is the border zone between the unaffected and the affected areas (p65 subunit, G). Cresyl violet staining demonstrated the penetration of macrophages in the center of the infarcted area (H). DOts show the center (inside area ~lfinner dots) and the penumbra (area between inner and outer dots) of the infarction. Scale bars= 25 p.m (A-F); 500”Mm (G and H).

K. Terai et al. /Brain Research 739 (1996) 343-349

infarcted areas of every case investigated. Astrocytes were not stained in the unaffected areas surrounding the lesions except for a single example where very weak staining was observed (Table 1). Fig. 2 shows the morphological features and localization of astrocytes stained for the p65 and p50 subunits of NF-KB in representative infarcted sites. The astrocytes were always of the reactive type (Fig. 2A–F) and were preferentially located in the penumbra, which is the border zone between areas affected by ischemia and unaffected areas (Fig. 2G). Cresyl violet staining demonstrated penetration of macrophages into the center of the infarcted area (Fig. 2H). In lesions of the third stage (Table 1), positive staining for both the p65 and p50 subunits of NF-KB was also detected in round cells, probably macrophages, distributed in affected areas. The number of positively stained astrocytes was small at this earlier stage (Fig. 3A–C). Vascular walls of small or medium size blood vessels were stained weakly, and were evenly distributed throughout the tissue except for infarcted areas (Fig. 3D). In such areas, the number of positively stained blood vessels was increased but the

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intensity of staining did not differ from unaffected areas (Table 1, cf. Fig. 3E,F) In this study, we used well-characterized antibodies against the p65 and p50 subunits of NF-K B [13,19,20,31,36] to determine its localization in infarcted areas of human brain. Prominent NF-KB staining was observed in the cytoplasm and processes of some astrocytes in the infarcted sites. This accumulation of NF-KB may reflect high activation or overexpression of NF-KB. A recent study using a ,transgenic mouse strain overexpressing the NF-KB p65 subunit indicated that cytoplasmic retention by IKB binding was the major in vivo mechanism for controlling the excess in overexpressing thymocytes [28]. The accumulation of NF-KB observed in our experiment may be caused by continuous induction of NF-KB expression by cytokines such as interleukin (IL)-2, which increases rnRNA levels of NF-KB subunits [1]. Some cytokines, including IL-2 and IL-6, have the ability to cause astrocytic proliferation and an increase of reactive astrogliosis [2]. The morphological features of astrocytes stained for NF-KB in the present

Fig. 3. Representative immunostaining for NF-KB p65 subunit of microphage-like cells and vascular walls. In the section shown in A, positively staining reactive astrocytes, as shown in the enlargement B, are rare (arrow). However, positively stained microphage-like cells are distributed in the infarcted area, as shown bv the arrowheads in the enlarged areas B and C. vascul~ wafls of small or medium size blood vessels ~e st~ned we*lY, ~d are even~Y distributed throughout the brain tissue (D) except for infarcted areas (E) where the number of positively stained blood vessels increases more than that in the unaffected area (F), but the intensity of staining is simil~ (cf. E and F). High power magnified regions (B,CEF) me shown as boxed ~eas on the low power photomicrographs (A,D). Scale bms = 2013pm (A); 50 km (B,Q,F); 500 wm (D)

34X

K. Terai et al. /Brain Research 739 (1996) 343-349

study always showed enlarged cell bodies and/or multiple long processes typical of reactive astrocytes. Interestingly, the expression of IL-6 in astrocytes seems to be involved in the activation of NF-KB [35]. These findings may indicate that a paracrine action of IL-6 in astrocytes causes self-proliferation, and that NF-KB is a necessary factor for such activity. Cytokines also have neurotrophic and/or angiogenic actions in vitro (see [12] for review). Astrocytes seem to be involved. For example, in an in vitro study, astrocytes were shown to produce and release IL-6 following treatment with IL- 1@or tumor necrosis factor-o- [24,25]. These cytokines are abundantly detected in ischemic brain areas [6,12]. Krupinski et al. showed that cerebral ischemic stroke causes active angiogenesis, which is more developed in the penumbra [16]. This might be a reason why astrocytes positively stained for NF-KB are preferentially located in the penumbra of the infarction. Macrophages may also produce cytokines to cause astrogliosis. The production of cytokines such as tumor necrosis factor-a-, IL- 1~ and IL-6 in lipopolysaccharidetreated macrophages was increased by inducing NF-KB activity [7, 15,41]. In our study, positively stained microphage-like cells were detected only in those infarction sites where the number of positive astrocytes was small. Therefore, NF-KB positive staining in macrophageIike cells might indicate a transient enhancement of NF-KB immunoreactivity at an early stage of ischemia, producing cytokines which later induced astrogliosis. A study of transient focal ischemia-reperfusion in the cerebral cortex indicated that the alternativeactivities of transcription factors, including NF-KB, is time-dependent [30]. Infiltration of leukocytes precedes the appearance of macrophages and astrogliosis [8]. The presence of adhesion molecules on endothelial cells seems to be of primary importance for stimulating infiltration of leukocytes into brain tissue damaged by ischemia. Administration of an antibody for intercellular adhesion molecule (ICAM) to the temporally aortic-occluded rabbit seemed to prevent the infiltration of leukocytes to the ischemic spinal cord thereby reducing the ischemic injury [9]. Many studies have suggested that the expression of ICAM-1, vascular cell adhesion molecule-1 [23] and E-selectin by endothelial cells is in response to NF-KB stimulation (see [10] for review). In this study there was no difference between the unaffected and affected infarction areas in the weak immunoreactivity for NF-KB in vascular walls. This result may suggest that enhancement of immunoreactivity does not appear in the endothelial cells or perhaps it appears only at some stage earlier than that investigated in this experiment.

Acknowledgements This research was supported by grants from the Jack Brown and Family Alzheimer Disease Research Fund, the

Alzheimer Society of British Columbia as well as donations from individual British Colombians.

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