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Inflammatory factors are elevated in brain microvessels in Alzheimer’s disease
Neurobiology of Aging 22 (2001) 837– 842
www.elsevier.com/locate/neuaging
Inflammatory factors are elevated in brain microvessels in Alzheimer’s disease Paula Grammas*, Roma Ovase Department of Pathology, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, Oklahoma City, OK 73104, USA Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, Oklahoma City, OK 73104, USA Received 9 February 2001; received in revised form 27 June 2001; accepted 2 July 2001
Abstract In Alzheimer’s disease (AD) inflammatory processes occur in pathologically vulnerable brain regions. The objective of this study is to compare both the release and the presence of microvessel-associated cytokines in vessels isolated from the brains of AD patients to microvessels from control brains. Microvessels are isolated from the cortices of AD patients and age-matched controls, without evidence of neurodegenerative disease. Inflammatory factors in the media are quantitated by ELISA and microvessel-associated mediators assessed by Western blot. Our results demonstrate that unstimulated AD microvessels release significantly higher levels of interleukin-1-(IL-1), IL-6, and tumor necrosis factor ␣ (TNF-␣) compared to non-AD microvessels. Levels of microvessel-associated monocyte chemoattractant protein (MCP-1) and IL-1 are high in AD-derived microvessels, but not detectable in non-AD microvessels. These results suggest that the cerebral microcirculation contributes inflammatory mediators to the milieu of the AD brain and may be involved in the pathogenesis of neuronal injury and death in this disorder. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Microvessels; Alzheimer’s disease; Inflammation; Cytokine
1. Introduction Despite a common clinical presentation (dementia) and similar neuropathology (plaques and tangles) Alzheimer’s disease (AD) is a heterogeneous disorder arising from multiple etiologies. A substantial literature demonstrates activation of inflammatory processes in pathologically vulnerable regions of the AD brain and documents the presence of a large number of inflammatory molecules [reviewed in 39]. Activated microglia are consistently associated with senile plaques in AD [33,34]. Amyloid beta (A) peptides, found in neuritic plaques, are likely to constitute a chronic inflammatory nidus, stimulating the actions of the complement system and pro-inflammatory cytokines [7,11,47]. The relevance of inflammatory processes to AD pathology is supported by animal studies showing that chronic infusion of lipopolysaccharide into the fourth ventricle of the rat brain reproduces many of the pathological changes observed in the AD brain [24]. Also, both in human and animal studies * Corresponding author. Tel.: ⫹1-405-271-3224; fax: ⫹1-405-2716437. E-mail address: [email protected] (P. Grammas).
anti-inflammatory drugs appear to reduce the risk of AD pathology and in some cases enhance cognitive performance [21,22,26,31,34]. Endogenous sources of cytokines and chemokines in the AD brain have begun to be defined. Activated microglia have been reported to produce IL-1, IL-6, TNF-␣, complement, and express monocyte chemoattractant protein 1 (MCP-1) [12,20,28,39,49]. Studies on astrocytes in AD show that these cells secrete IL-1, IL-6, and transforming growth factor  (TGF) [1,8,39,44,48]. Much less attention has focused on the ability of the brain microvasculature to either produce or express inflammatory mediators in AD. Suo et al. [46] demonstrate that in human endothelial cells A induces an inflammatory cascade that includes secretion of interferon ␥ (IFN-␥), IL-1 and expression of CD40. Another inflammatory molecule the intercellular adhesion molecule-1 (ICAM-1), a surface glycoprotein expressed in inflammation, has been demonstrated on brain endothelial cells in AD [13]. Previous work from our laboratory demonstrates that in the AD brain endothelial cells are “activated” and express the inflammatory mediator CAP37 (cationic antimicrobial protein, MW 37 kDa) on their surface [40]. We have also shown a significant over expression of induc-
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ible nitric oxide synthase (NOS) in microvessels isolated from AD brains [10]. This NOS isoform is not usually present in endothelial cells, but its expression can be induced in endothelial cells that are activated by inflammatory stimulation [19]. Taken together, these data implicate the cerebral microvasculature as a source of inflammatory factors in AD. The objective of this study is to examine both the release of soluble inflammatory mediators and the presence of microvessel-associated inflammatory mediators in vessels isolated from AD brains and compare these data to those obtained in microvessels isolated from age-matched control patients.
2. Materials and methods 2.1. Human microvessel isolation Human autopsy brains were obtained approximately 4 to 15 h postmortem and frozen at -70°C until dissection. The clinical diagnosis of primary AD was confirmed by neuropathological examination. Control samples from agematched patients without evidence of neuropathology were also collected. The age range for AD (69 – 86 years) and control samples (66 –75 years) are similar. Microvessels were isolated from pooled temporal, parietal, and frontal cortices, as we have previously described [4,17,18]. Briefly, cortices were placed in cold Hank’s balanced salt solution (HBSS), scissor minced, and homogenized using 20 up-anddown strokes in a Teflon glass homogenizer. After centrifugation (3000 g) for 15 min at 4°C, the supernatant was discarded and the pellet resuspended in cold HBSS containing 15% dextran and 5% fetal calf serum (FCS). The suspension was then centrifuged at 5500 g for 20 min at 4°C. The supernatant was again discarded and the pellet filtered through a 210 m nylon mesh. The resulting filtrate was then collected off a 53 m nylon mesh sieve. Microvessels were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS and dimethyl sulfoxide and stored frozen in liquid nitrogen until use. This procedure yields approximately 6 to 10 mg microvessel protein from 15 g of human cortex. A separate microvessel preparation was isolated from each human brain. The purity of the microvessel preparations was routinely monitored by phasecontrast microscopy. 2.2. Preparation of microvessel conditioned media and lysates Microvessels were quick-thawed at 37°C and centrifuged at 2000 g for 15 min. Microvessels were washed three times with cold Hank’s balanced salt solution and resuspended in serum-free DMEM containing 1% lactalbumin hydrogenate (LAH). Microvessels (⬃50 g/sample) were incubated in 1 ml for 4 to 6 h at 37°C in a 95% O2/5% O2 incubator, and
then centrifuged (2000 g). The media were collected and used for ELISA. To determine microvessel-associated inflammatory mediators microvessels were lysed using 2% SDS containing EDTA (0.5M), and lysates (50 g/lane) used for Western blots analysis. 2.3. Cytokine analysis using ELISA Levels of IL-1, IL-6 and TNF-␣ in conditioned media from AD and non-AD samples were measured by ELISA. ELISA kits were obtained from Boehringer Mannheim. Levels of biologically active IL-6, IL-1, and TNF-␣ were determined using a color-based assay. In this assay the samples were bound by a biotin-labeled antibody and the peroxidase conjugated detection antibody. This complex bound via the biotin-labeled antibody to the streptavidincoated microtiter plate. The plate was incubated at room temperature for 2 h on an orbital shaker (⬃250 rpm). Next, the plate was washed and bound peroxidase was developed by adding tetramethylbenzidine as a substrate. The developed color (read at 450 nm) was proportional to the concentration of these cytokines. This ELISA system detects both natural and recombinant human IL-1 and IL-6, and TNF-␣ has a measuring range between 5–700 pg/ml. All samples measured within the range of the standard curve. 2.4. MCP-1 and IL-1 detection by Western blot Microvessels lysates (50 g/lane) were loaded for SDS polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane for Western blot analyses. The blots were washed two times using Tris Buffered Saline with Tween 20 (TBST), incubated in milk solution (TBST with 3% non-fat dry milk) for 1 h, and washed two times with TBST. The blots were then incubated in the primary antibody (MCP-1: R&D systems, catalog # AF 279-NA; IL-1: Santa Cruz Biotechnology, catalog # sc-7884) in milk solution (dilution 1:500) for 1 h, washed 3 times with TBST, incubated with the secondary antibody (Sigma, A-8062 for MCP-1 and Bio-Rad, 1706515 for IL-1) in milk solution (dilution 1: 4000) for 1 h, and washed 3 times with TBST. The blots were then enhanced (Bio-Rad Immun-Star Kit, catalog #170 –5011) and visualized on film, and the optical density of bands quantitated using a GEL DOC 2000.
3. Results 3.1. Both microvessel-associated and soluble IL-1 are elevated in AD microvessels AD and control microvessels were analyzed for the release of soluble IL-1 and the presence of microvesselassociated IL-1. Incubation of isolated microvessels in serum-free medium resulted in a significantly (P ⬍ 0.01)
P. Grammas, R. Ovase / Neurobiology of Aging 22 (2001) 837– 842
Fig. 1. IL-1 release is higher in AD microvessels compared to microvessels from control brain. Microvessels were washed and incubated for 6 h with serum free DMEM containing 1% LAH (n ⫽ 9). Microvessels were centrifuged and the supernatant assayed for IL- using an ELISA assay. *P ⬍ 0.01, significantly different from non-AD vessels.
higher (60%) release of IL-1 into the media by ADderived microvessels compared to control-derived vessels (Fig 1). Western blot analysis of microvessel-associated IL-1 showed strong reactivity of AD-derived vessels to IL-1 antibody with no detectable reactivity in control microvessels (Fig. 2). 3.2. Soluble IL-6 levels are higher in AD-derived microvessels compared to control-derived microvessels There was a significantly (P ⬍ 0.05) higher level of IL-6 produced by AD vessels (80%) under, basal unstimulated conditions compared to control-derived microvessels (Fig. 3). Microvessel-associated IL-6 was not detectable in either AD-derived or control derived microvessels (data not shown). 3.3. Release of TNF-␣ is higher in AD microvessels compared to microvessels from control-derived brains Incubation of isolated microvessels in serum-free medium resulted in a significantly (P ⬍ 0.02) higher (88%) release of TNF-␣ into the media by AD vessels compared to control-derived microvessels (Fig. 4). Microvessel-associated TNF-␣ was not detectable in either AD-derived or control derived microvessels (data not shown). 3.4. Microvessel-associated MCP-1 is elevated in AD Western blot analysis using antibodies to human MCP-1 showed a high level of reactivity to AD-derived microvessel
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Fig. 2. Microvessel-associated IL-1 is elevated in AD. Microvessels lysates (50 g/lane) were loaded for SDS-PAGE and Western blot analysis performed using antibodies to IL-1. Unstimulated AD microvessels (lanes 1,2), and non-AD vessels (lanes 3,4). These blots are representative of 15 separate experiments showing similar results.
lysates and no detectable reactivity in non-AD control microvessels (Fig. 5).
4. Discussion In this study we demonstrate that AD microvessels release significantly higher levels of IL-1, IL-6, and TNF-␣ under basal, unstimulated conditions, compared to non-AD derived microvessels. High levels of microvessel-associated MCP-1 and IL-1, as determined by Western blots, are detectable in AD but not in non-AD microvessels. These data demonstrate elevated levels of inflammatory mediators in isolated AD brain microvessels, and suggest that the cerebral microcirculation contributes noxious mediators to the milieu of the AD brain and plays a role in the pathogenesis of neuronal injury in this disorder. Although increasing evidence suggests inflammatory mechanisms are active in the AD brain the identity of the mediators and cell types involved have not been clarified. It is likely that spatial-temporal interactions and/or synergy among non-neuronal cell types and soluble factors occur and that the cerebral microvasculature is an important participant in these processes. For example, A 25–35 in the presence of IL-1, a cytokine released by astrocytes, microglia and endothelial cells, causes production of nitric oxide in the C6 astrocytic cell line [43]. A fibrils dosedependently induce release of nitric oxide in vivo from microglia [27,35]. A similar mechanism may be operative in brain microvessels isolated from AD patients that are heavily invested with amyloid and, as we have previously shown, produce high levels of nitric oxide [10]. Finally, in
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Fig. 3. IL-6 release is higher in AD microvessels compared to microvessels from control brain. Microvessels were washed and incubated for 6 h with serum free DMEM containing 1% LAH. Microvessels were centrifuged and the supernatant assayed for IL-6 using an ELISA assay. *P ⬍ 0.05, significantly different from non-AD vessels.
Fig. 4. TNF-␣ release is higher in AD microvessels compared to microvessels from control brain. Microvessels were washed and incubated for 6 h with serum free DMEM containing 1% LAH. Microvessels were centrifuged and the supernatant assayed for TNF-␣ using an ELISA assay. *P ⬍ 0.02, significantly different from non-AD vessels.
human endothelial cells A causes release of IL-1 and IL-1 upregulates mRNA for amyloid precursor protein [14, 16,46], implicating this cytokine in a positive inflammatory feedback loop in the vasculature that leads to production of neurotoxic factors, such as A. Endothelial cells appear to be a rich source of both cytokines and chemokines [15]. Furthermore, release of these inflammatory factors from the endothelium occurs in response to a wide variety of stimuli. For example, human umbilical vein endothelial cells produce IL-6 in response to thrombin, lipopolysaccharide and TNF-␣ [6]. Histamine, long known increase vascular permeability, has been shown to induce synthesis of IL-6 and IL-8 in human coronary artery endothelial cells [30]. Both homocysteine and endothelin-1 induce expression of MCP-1 in endothelial cells [5,41]. In addition, viral infection causes release of cytokines by endothelial cells [23]. These data are relevant for AD because many of the mediators that evoke endothelial cell cytokine release (e.g. thrombin, homocysteine and endothelin-1) have also been implicated in the pathogenesis of AD [2,36 –37]. It is clear soluble inflammatory factors are important since virtually all cytokines and chemokines that have been studied in AD including IL-1, IL-6, TNF-␣, IL-8, TGF- and MIP-1␣ are upregulated [39]. In addition to these soluble mediators cell surface molecules are important in regulating the inflammatory response in the brain. In human brain endothelial cells TNF-␣ induces expression of the cell adhesion molecules ICAM-1 and VCAM-1 [9,29]. In the AD brain there is elevated expression of ICAM-1 in the cerebral microcirculation [13]. In this regard, here we demonstrate, by Western blot analysis, strong expression of the monocyte chemoattractant protein MCP-1 in isolated AD but not control vessels. Our results are also supported by the
findings of Hofman et al. [25] who recently demonstrated MCP-1 expression in the cerebral microcirculation in tissue sections from AD but not control patient brains. We have previously shown that the cerebral microcirculation from AD patients expresses CAP37, an inflammatory protein that stimulates monocyte adhesion to endothelial cells and monocyte activation [40]. These data, in conjunction with our present results, highlight a central role for the cerebral endothelium as a regulator of white blood cells transgression across the blood brain barrier as well as a mediator of inflammatory events within the CNS. Our data, and a rapidly growing literature, confirm that the AD brain is in a pro-inflammatory state. The central question to be addressed is inflammation causal to neurodegeneration in AD or inflammatory processes secondarily activated only to “clean up” the tissue destruction evoked by other mechanisms? Although the temporal expression of inflammatory factors in relation to neuronal loss in AD is difficult to discern using end-stage AD post-mortem tissues, a large body of evidence supports a pathophysiologic role for inflammation in the neuronal cell death characteristic of AD [39]. For example, inflammatory mediators co-localize in the AD brain with those regions that exhibit high levels of AD pathology and are most highly expressed in the vicinity of A deposits and neurofibrillary tangles [32]. Clinical studies have provided evidence that non-steroidal anti-inflammatory drugs are beneficial in preventing and/or alleviating some of AD symptoms [34,42]. Our data showing an upregulation of vascular-derived inflammatory mediators in AD is significant because of the topographic association of capillaries with neuritic plaques and the colocalization of vascular-derived heparan sulfate proteoglycan deposits with senile plaques [3,38].
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References
Fig. 5. Microvessel-associated MCP-1 is elevated in AD. Microvessels lysates (50 g/lane) were loaded for SDS-PAGE and Western blot analysis performed using antibodies to MCP-1. Unstimulated AD microvessels (lanes 1,2), and non-AD vessels (lane 3,4). These blots are representative of 8 separate experiments showing similar results.
Although endothelial cells produce relatively small amounts of cytokines compared to astrocytes and microglia, because the brain endothelium is continuously exposed to potentially noxious elements in the blood this cell type is uniquely positioned to initiate a destructive inflammatory cycle. Thus, release of inflammatory mediators from a dysfunctional endothelium in AD could, via paracrine stimulation of other non-neuronal cell types, lead to increased levels of inflammatory mediators, reactive oxygen species and other neurotoxic molecules in the brain. Our data implicate the cerebral microcirculation as an important source of inflammatory mediators and a key regulator facilitating the passage of inflammatory cells into the brain in AD. These data as well as reports that suggest a “link” between cardiovascular disease (e.g. atherosclerosis) and the development of AD [45] suggest that the vasculature is intimately involved in the development of AD and is an unexplored target for therapeutic intervention.
Acknowledgments The author gratefully acknowledges the secretarial assistance of Theresa Rush and Mel Beery. This work was supported in part by a Zenith Award from the Alzheimer’s Association (PG), and NIH grant RO1 AG 15964 (PG). Dr. Grammas is the recipient of the Alfred M. Shideler Professorship in Pathology.
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Inflammatory factors are elevated in brain microvessels in Alzheimer’s disease Paula Grammas*, Roma Ovase Department of Pathology, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, Oklahoma City, OK 73104, USA Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, Oklahoma City, OK 73104, USA Received 9 February 2001; received in revised form 27 June 2001; accepted 2 July 2001
Abstract In Alzheimer’s disease (AD) inflammatory processes occur in pathologically vulnerable brain regions. The objective of this study is to compare both the release and the presence of microvessel-associated cytokines in vessels isolated from the brains of AD patients to microvessels from control brains. Microvessels are isolated from the cortices of AD patients and age-matched controls, without evidence of neurodegenerative disease. Inflammatory factors in the media are quantitated by ELISA and microvessel-associated mediators assessed by Western blot. Our results demonstrate that unstimulated AD microvessels release significantly higher levels of interleukin-1-(IL-1), IL-6, and tumor necrosis factor ␣ (TNF-␣) compared to non-AD microvessels. Levels of microvessel-associated monocyte chemoattractant protein (MCP-1) and IL-1 are high in AD-derived microvessels, but not detectable in non-AD microvessels. These results suggest that the cerebral microcirculation contributes inflammatory mediators to the milieu of the AD brain and may be involved in the pathogenesis of neuronal injury and death in this disorder. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Microvessels; Alzheimer’s disease; Inflammation; Cytokine
1. Introduction Despite a common clinical presentation (dementia) and similar neuropathology (plaques and tangles) Alzheimer’s disease (AD) is a heterogeneous disorder arising from multiple etiologies. A substantial literature demonstrates activation of inflammatory processes in pathologically vulnerable regions of the AD brain and documents the presence of a large number of inflammatory molecules [reviewed in 39]. Activated microglia are consistently associated with senile plaques in AD [33,34]. Amyloid beta (A) peptides, found in neuritic plaques, are likely to constitute a chronic inflammatory nidus, stimulating the actions of the complement system and pro-inflammatory cytokines [7,11,47]. The relevance of inflammatory processes to AD pathology is supported by animal studies showing that chronic infusion of lipopolysaccharide into the fourth ventricle of the rat brain reproduces many of the pathological changes observed in the AD brain [24]. Also, both in human and animal studies * Corresponding author. Tel.: ⫹1-405-271-3224; fax: ⫹1-405-2716437. E-mail address: [email protected] (P. Grammas).
anti-inflammatory drugs appear to reduce the risk of AD pathology and in some cases enhance cognitive performance [21,22,26,31,34]. Endogenous sources of cytokines and chemokines in the AD brain have begun to be defined. Activated microglia have been reported to produce IL-1, IL-6, TNF-␣, complement, and express monocyte chemoattractant protein 1 (MCP-1) [12,20,28,39,49]. Studies on astrocytes in AD show that these cells secrete IL-1, IL-6, and transforming growth factor  (TGF) [1,8,39,44,48]. Much less attention has focused on the ability of the brain microvasculature to either produce or express inflammatory mediators in AD. Suo et al. [46] demonstrate that in human endothelial cells A induces an inflammatory cascade that includes secretion of interferon ␥ (IFN-␥), IL-1 and expression of CD40. Another inflammatory molecule the intercellular adhesion molecule-1 (ICAM-1), a surface glycoprotein expressed in inflammation, has been demonstrated on brain endothelial cells in AD [13]. Previous work from our laboratory demonstrates that in the AD brain endothelial cells are “activated” and express the inflammatory mediator CAP37 (cationic antimicrobial protein, MW 37 kDa) on their surface [40]. We have also shown a significant over expression of induc-
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ible nitric oxide synthase (NOS) in microvessels isolated from AD brains [10]. This NOS isoform is not usually present in endothelial cells, but its expression can be induced in endothelial cells that are activated by inflammatory stimulation [19]. Taken together, these data implicate the cerebral microvasculature as a source of inflammatory factors in AD. The objective of this study is to examine both the release of soluble inflammatory mediators and the presence of microvessel-associated inflammatory mediators in vessels isolated from AD brains and compare these data to those obtained in microvessels isolated from age-matched control patients.
2. Materials and methods 2.1. Human microvessel isolation Human autopsy brains were obtained approximately 4 to 15 h postmortem and frozen at -70°C until dissection. The clinical diagnosis of primary AD was confirmed by neuropathological examination. Control samples from agematched patients without evidence of neuropathology were also collected. The age range for AD (69 – 86 years) and control samples (66 –75 years) are similar. Microvessels were isolated from pooled temporal, parietal, and frontal cortices, as we have previously described [4,17,18]. Briefly, cortices were placed in cold Hank’s balanced salt solution (HBSS), scissor minced, and homogenized using 20 up-anddown strokes in a Teflon glass homogenizer. After centrifugation (3000 g) for 15 min at 4°C, the supernatant was discarded and the pellet resuspended in cold HBSS containing 15% dextran and 5% fetal calf serum (FCS). The suspension was then centrifuged at 5500 g for 20 min at 4°C. The supernatant was again discarded and the pellet filtered through a 210 m nylon mesh. The resulting filtrate was then collected off a 53 m nylon mesh sieve. Microvessels were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS and dimethyl sulfoxide and stored frozen in liquid nitrogen until use. This procedure yields approximately 6 to 10 mg microvessel protein from 15 g of human cortex. A separate microvessel preparation was isolated from each human brain. The purity of the microvessel preparations was routinely monitored by phasecontrast microscopy. 2.2. Preparation of microvessel conditioned media and lysates Microvessels were quick-thawed at 37°C and centrifuged at 2000 g for 15 min. Microvessels were washed three times with cold Hank’s balanced salt solution and resuspended in serum-free DMEM containing 1% lactalbumin hydrogenate (LAH). Microvessels (⬃50 g/sample) were incubated in 1 ml for 4 to 6 h at 37°C in a 95% O2/5% O2 incubator, and
then centrifuged (2000 g). The media were collected and used for ELISA. To determine microvessel-associated inflammatory mediators microvessels were lysed using 2% SDS containing EDTA (0.5M), and lysates (50 g/lane) used for Western blots analysis. 2.3. Cytokine analysis using ELISA Levels of IL-1, IL-6 and TNF-␣ in conditioned media from AD and non-AD samples were measured by ELISA. ELISA kits were obtained from Boehringer Mannheim. Levels of biologically active IL-6, IL-1, and TNF-␣ were determined using a color-based assay. In this assay the samples were bound by a biotin-labeled antibody and the peroxidase conjugated detection antibody. This complex bound via the biotin-labeled antibody to the streptavidincoated microtiter plate. The plate was incubated at room temperature for 2 h on an orbital shaker (⬃250 rpm). Next, the plate was washed and bound peroxidase was developed by adding tetramethylbenzidine as a substrate. The developed color (read at 450 nm) was proportional to the concentration of these cytokines. This ELISA system detects both natural and recombinant human IL-1 and IL-6, and TNF-␣ has a measuring range between 5–700 pg/ml. All samples measured within the range of the standard curve. 2.4. MCP-1 and IL-1 detection by Western blot Microvessels lysates (50 g/lane) were loaded for SDS polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane for Western blot analyses. The blots were washed two times using Tris Buffered Saline with Tween 20 (TBST), incubated in milk solution (TBST with 3% non-fat dry milk) for 1 h, and washed two times with TBST. The blots were then incubated in the primary antibody (MCP-1: R&D systems, catalog # AF 279-NA; IL-1: Santa Cruz Biotechnology, catalog # sc-7884) in milk solution (dilution 1:500) for 1 h, washed 3 times with TBST, incubated with the secondary antibody (Sigma, A-8062 for MCP-1 and Bio-Rad, 1706515 for IL-1) in milk solution (dilution 1: 4000) for 1 h, and washed 3 times with TBST. The blots were then enhanced (Bio-Rad Immun-Star Kit, catalog #170 –5011) and visualized on film, and the optical density of bands quantitated using a GEL DOC 2000.
3. Results 3.1. Both microvessel-associated and soluble IL-1 are elevated in AD microvessels AD and control microvessels were analyzed for the release of soluble IL-1 and the presence of microvesselassociated IL-1. Incubation of isolated microvessels in serum-free medium resulted in a significantly (P ⬍ 0.01)
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Fig. 1. IL-1 release is higher in AD microvessels compared to microvessels from control brain. Microvessels were washed and incubated for 6 h with serum free DMEM containing 1% LAH (n ⫽ 9). Microvessels were centrifuged and the supernatant assayed for IL- using an ELISA assay. *P ⬍ 0.01, significantly different from non-AD vessels.
higher (60%) release of IL-1 into the media by ADderived microvessels compared to control-derived vessels (Fig 1). Western blot analysis of microvessel-associated IL-1 showed strong reactivity of AD-derived vessels to IL-1 antibody with no detectable reactivity in control microvessels (Fig. 2). 3.2. Soluble IL-6 levels are higher in AD-derived microvessels compared to control-derived microvessels There was a significantly (P ⬍ 0.05) higher level of IL-6 produced by AD vessels (80%) under, basal unstimulated conditions compared to control-derived microvessels (Fig. 3). Microvessel-associated IL-6 was not detectable in either AD-derived or control derived microvessels (data not shown). 3.3. Release of TNF-␣ is higher in AD microvessels compared to microvessels from control-derived brains Incubation of isolated microvessels in serum-free medium resulted in a significantly (P ⬍ 0.02) higher (88%) release of TNF-␣ into the media by AD vessels compared to control-derived microvessels (Fig. 4). Microvessel-associated TNF-␣ was not detectable in either AD-derived or control derived microvessels (data not shown). 3.4. Microvessel-associated MCP-1 is elevated in AD Western blot analysis using antibodies to human MCP-1 showed a high level of reactivity to AD-derived microvessel
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Fig. 2. Microvessel-associated IL-1 is elevated in AD. Microvessels lysates (50 g/lane) were loaded for SDS-PAGE and Western blot analysis performed using antibodies to IL-1. Unstimulated AD microvessels (lanes 1,2), and non-AD vessels (lanes 3,4). These blots are representative of 15 separate experiments showing similar results.
lysates and no detectable reactivity in non-AD control microvessels (Fig. 5).
4. Discussion In this study we demonstrate that AD microvessels release significantly higher levels of IL-1, IL-6, and TNF-␣ under basal, unstimulated conditions, compared to non-AD derived microvessels. High levels of microvessel-associated MCP-1 and IL-1, as determined by Western blots, are detectable in AD but not in non-AD microvessels. These data demonstrate elevated levels of inflammatory mediators in isolated AD brain microvessels, and suggest that the cerebral microcirculation contributes noxious mediators to the milieu of the AD brain and plays a role in the pathogenesis of neuronal injury in this disorder. Although increasing evidence suggests inflammatory mechanisms are active in the AD brain the identity of the mediators and cell types involved have not been clarified. It is likely that spatial-temporal interactions and/or synergy among non-neuronal cell types and soluble factors occur and that the cerebral microvasculature is an important participant in these processes. For example, A 25–35 in the presence of IL-1, a cytokine released by astrocytes, microglia and endothelial cells, causes production of nitric oxide in the C6 astrocytic cell line [43]. A fibrils dosedependently induce release of nitric oxide in vivo from microglia [27,35]. A similar mechanism may be operative in brain microvessels isolated from AD patients that are heavily invested with amyloid and, as we have previously shown, produce high levels of nitric oxide [10]. Finally, in
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Fig. 3. IL-6 release is higher in AD microvessels compared to microvessels from control brain. Microvessels were washed and incubated for 6 h with serum free DMEM containing 1% LAH. Microvessels were centrifuged and the supernatant assayed for IL-6 using an ELISA assay. *P ⬍ 0.05, significantly different from non-AD vessels.
Fig. 4. TNF-␣ release is higher in AD microvessels compared to microvessels from control brain. Microvessels were washed and incubated for 6 h with serum free DMEM containing 1% LAH. Microvessels were centrifuged and the supernatant assayed for TNF-␣ using an ELISA assay. *P ⬍ 0.02, significantly different from non-AD vessels.
human endothelial cells A causes release of IL-1 and IL-1 upregulates mRNA for amyloid precursor protein [14, 16,46], implicating this cytokine in a positive inflammatory feedback loop in the vasculature that leads to production of neurotoxic factors, such as A. Endothelial cells appear to be a rich source of both cytokines and chemokines [15]. Furthermore, release of these inflammatory factors from the endothelium occurs in response to a wide variety of stimuli. For example, human umbilical vein endothelial cells produce IL-6 in response to thrombin, lipopolysaccharide and TNF-␣ [6]. Histamine, long known increase vascular permeability, has been shown to induce synthesis of IL-6 and IL-8 in human coronary artery endothelial cells [30]. Both homocysteine and endothelin-1 induce expression of MCP-1 in endothelial cells [5,41]. In addition, viral infection causes release of cytokines by endothelial cells [23]. These data are relevant for AD because many of the mediators that evoke endothelial cell cytokine release (e.g. thrombin, homocysteine and endothelin-1) have also been implicated in the pathogenesis of AD [2,36 –37]. It is clear soluble inflammatory factors are important since virtually all cytokines and chemokines that have been studied in AD including IL-1, IL-6, TNF-␣, IL-8, TGF- and MIP-1␣ are upregulated [39]. In addition to these soluble mediators cell surface molecules are important in regulating the inflammatory response in the brain. In human brain endothelial cells TNF-␣ induces expression of the cell adhesion molecules ICAM-1 and VCAM-1 [9,29]. In the AD brain there is elevated expression of ICAM-1 in the cerebral microcirculation [13]. In this regard, here we demonstrate, by Western blot analysis, strong expression of the monocyte chemoattractant protein MCP-1 in isolated AD but not control vessels. Our results are also supported by the
findings of Hofman et al. [25] who recently demonstrated MCP-1 expression in the cerebral microcirculation in tissue sections from AD but not control patient brains. We have previously shown that the cerebral microcirculation from AD patients expresses CAP37, an inflammatory protein that stimulates monocyte adhesion to endothelial cells and monocyte activation [40]. These data, in conjunction with our present results, highlight a central role for the cerebral endothelium as a regulator of white blood cells transgression across the blood brain barrier as well as a mediator of inflammatory events within the CNS. Our data, and a rapidly growing literature, confirm that the AD brain is in a pro-inflammatory state. The central question to be addressed is inflammation causal to neurodegeneration in AD or inflammatory processes secondarily activated only to “clean up” the tissue destruction evoked by other mechanisms? Although the temporal expression of inflammatory factors in relation to neuronal loss in AD is difficult to discern using end-stage AD post-mortem tissues, a large body of evidence supports a pathophysiologic role for inflammation in the neuronal cell death characteristic of AD [39]. For example, inflammatory mediators co-localize in the AD brain with those regions that exhibit high levels of AD pathology and are most highly expressed in the vicinity of A deposits and neurofibrillary tangles [32]. Clinical studies have provided evidence that non-steroidal anti-inflammatory drugs are beneficial in preventing and/or alleviating some of AD symptoms [34,42]. Our data showing an upregulation of vascular-derived inflammatory mediators in AD is significant because of the topographic association of capillaries with neuritic plaques and the colocalization of vascular-derived heparan sulfate proteoglycan deposits with senile plaques [3,38].
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References
Fig. 5. Microvessel-associated MCP-1 is elevated in AD. Microvessels lysates (50 g/lane) were loaded for SDS-PAGE and Western blot analysis performed using antibodies to MCP-1. Unstimulated AD microvessels (lanes 1,2), and non-AD vessels (lane 3,4). These blots are representative of 8 separate experiments showing similar results.
Although endothelial cells produce relatively small amounts of cytokines compared to astrocytes and microglia, because the brain endothelium is continuously exposed to potentially noxious elements in the blood this cell type is uniquely positioned to initiate a destructive inflammatory cycle. Thus, release of inflammatory mediators from a dysfunctional endothelium in AD could, via paracrine stimulation of other non-neuronal cell types, lead to increased levels of inflammatory mediators, reactive oxygen species and other neurotoxic molecules in the brain. Our data implicate the cerebral microcirculation as an important source of inflammatory mediators and a key regulator facilitating the passage of inflammatory cells into the brain in AD. These data as well as reports that suggest a “link” between cardiovascular disease (e.g. atherosclerosis) and the development of AD [45] suggest that the vasculature is intimately involved in the development of AD and is an unexplored target for therapeutic intervention.
Acknowledgments The author gratefully acknowledges the secretarial assistance of Theresa Rush and Mel Beery. This work was supported in part by a Zenith Award from the Alzheimer’s Association (PG), and NIH grant RO1 AG 15964 (PG). Dr. Grammas is the recipient of the Alfred M. Shideler Professorship in Pathology.
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