© 1999 American Heart Association, Inc.
Original Contributions |
Tissue-Destructive Macrophages in Giant Cell Arteritis
From the Department of Medicine (H.L.R., M.K, A.B., J.J.G., C.M.W.), Division of Rheumatology, Mayo Clinic, Rochester, Minn, and the Department of Physiology and Biophysics (L.I.S.), Case Western Reserve University, Cleveland, Ohio.
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Abstract |
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Abstract—Giant cell arteritis (GCA) is an inflammatory vasculopathy in which T cells and macrophages infiltrate the wall of medium and large arteries. Clinical consequences such as blindness and stroke are related to arterial occlusion. Formation of aortic aneurysms may result from necrosis of smooth muscle cells and fragmentation of elastic membranes. The molecular mechanisms of arterial wall injury in GCA are not understood. To identify mechanisms of arterial damage, gene expression in inflamed and unaffected temporal artery specimens was compared by differential display polymerase chain reaction. Genes differentially expressed in arterial lesions included 3 products encoded by the mitochondrial genome. Immunohistochemistry with antibodies specific for a 65-kDa mitochondrial antigen revealed that increased expression of mitochondrial products was characteristic of multinucleated giant cells and of CD68+ macrophages that cluster in the media and at the media-intima junction. 4-Hydroxy-2-nonenal adducts, products of lipid peroxidation, were detected on smooth muscle cells and on tissue infiltrating cells, in close proximity to multinucleated giant cells and CD68+ macrophages. Also, giant cells and macrophages with overexpression of mitochondrial products were able to synthesize metalloproteinase-2. Our data suggest that in the vascular lesions characteristic for GCA, a subset of macrophages has the potential to support several pathways of arterial injury, including the release of reactive oxygen species and the production of metalloproteinase-2. This macrophage subset is topographically defined and is also identified by overexpression of mitochondrial genes. Because these macrophages have a high potential to promote several mechanisms of arterial wall damage, they should be therapeutically targeted to prevent blood vessel destruction.
Key Words: reactive oxygen species • vasculitis • metalloproteinase • macrophage
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Introduction |
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Giant cell arteritis (GCA) is an inflammatory vasculopathy that affects mainly the upper extremity and extracranial branches of the aorta.1 2 The disease preferentially involves medium-sized arteries, but aortitis may occur more frequently than previously thought.3 Emerging data support the concept that GCA represents the sequelae of an immune response directed against antigens that reside in the arterial wall.
Evidence has been collected that T lymphocytes play a critical role in
the disease process.4 Curiously, IFN-
as a key T-cell
product accumulates in the adventitia of affected blood
vessels.5 T cells that produce IFN- intermingle with a
functionally defined subset of macrophages that produces
IL-1ß, IL-6, and TGF-ß.6 7 The topographical
arrangement of activated T cells and macrophages that
produce cytokines indicates that the adventitia is the site of
the initial immunologic injury. However, tissue-destructive events in
this arteritis are focused on the media, media-intima junction, and
intima. The arterial injury either results in intimal
hyperplasia with subsequent luminal occlusion or in wall fragmentation
with aneurysm formation. The relationship between the events
that occur in the adventitia and the tissue destruction that affects
the media and intima has not been addressed.
Presently, little information exists on the molecular mechanisms that culminate in arterial damage. Proteolytic enzymes have been detected in the infiltrate. Matrix metalloproteinase (MMP)–2 and MMP-9 have both been described to be expressed in tissue-infiltrating inflammatory cells and possibly in cellular components of the arterial wall.6 8 However, MMPs are produced as proenzymes, and how they are activated in the arterial wall to exhibit proteolytic and injurious activity is not clear.9 Evidence also exists that nitric oxide (NO) is produced in the arterial tissue. Macrophages that reside in the intima express high levels of inducible nitric oxide synthase (iNOS) and are likely to produce this toxic mediator.6 The presence of NO has been associated with proinflammatory and tissue-damaging effects.10 However, the spatial restriction of macrophages that express iNOS in the intima suggests that NO does not contribute to media destruction.
To identify mechanisms involved in the destruction of the medial layer and the elastic membranes of the artery, we compared gene expression in inflamed and nonaffected temporal arteries by differential display polymerase chain reaction (DD-PCR).11 The power of this technology relates to its ability to provide information on the upregulation of gene transcripts derived from either infiltrating or residing cells, which allows for the detection and characterization of novel pathways involved in the disease process. Three of 19 gene products overrepresented in affected arterial tissue were derived from the mitochondrial genome. Increased transcription of mitochondrial gene products led to the recognition of mitochondrial activation in some but not all cells in the vascular lesions. Multinucleated giant cells and macrophages accumulated in the media, clustered along the elastic laminae, and situated in the center of tissue damage overexpressed mitochondrial genes. To examine the destructive potential of these particular macrophages, evidence was sought for the action of reactive oxygen species (ROS) and the expression of MMP in the arterial wall. Because free radicals have a limited life span in the tissue, they are difficult to identify directly. The most susceptible components of oxidative damage are polyunsaturated fatty acids, and the major products of lipid peroxidation are 4-hydroxy-2-nonenal (HNE) adducts.12 HNE adducts could be readily detected in temporal arteries with vasculitic lesions. Macrophages with upregulated mitochondrial activity and the formation of HNE adducts spatially coincided around the elastic membranes and in the media. Additional characterization of this macrophage subset revealed that the upregulation of mitochondrial activity and the formation of HNE adducts closely correlated with the expression of MMP-2. We propose that this macrophage subpopulation, which includes multinucleated giant cells, is a major effector of tissue injury in GCA.
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Materials and Methods |
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Samples
Temporal artery specimens were obtained during routine diagnostic biopsies. Ten patients with histologic signs of GCA were enrolled in the study after written, informed consent was obtained. Temporal artery samples that were histomorphologically negative and were derived from patients that lacked clinical evidence of systemic vasculitis served as controls. These patients underwent temporal artery biopsy as part of an evaluation for fever of unknown origin or unexplained constitutional symptoms. Samples were immediately snap-frozen in liquid nitrogen until used for RNA isolation. For immunohistochemistry, tissue embedded in OCT or paraffin from 15 patients with GCA was used.
Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Paque (Amersham Biotech/Pharmacia). Cells were stimulated with 1 µg/mL phorbol 12-myristate 13-acetate, 3.3 µmol/L ionomycin, and 1 µg/mL LPS for 4 to 6 hours (Sigma Chemical Co).
Differential Display Polymerase Chain Reaction
Gene expression in temporal artery specimens of GCA, control
patients, and activated PBMCs was analyzed by DD-PCR.
Empirically, 4 unique oligo dT primers and 20 random primers can
amplify the entire mRNA repertoire under nonstringent PCR
conditions.11 For the present study, 4 oligo dT
(T12V A, C, G, or T) and 10 random primers
(random A 5'-CTG ATC CAT G-3'; random B 5'-CTT GAT TGC C-3'; random C
5'-CTG CTC TCA G-3'; random D 5'-CGA TGC TTG A-3'; random E 5'-TCC AGT
ATG G-3'; random F 5'-ACG TCA TGA C-3'; random G 5'-GTA CTC AGA C-3';
random H 5'-TGG ACT CTC A-3'; random I 5'-GTC ATC ATC G-3'; and random
J 5'-CAT GTC AAG C-3') were arbitrarily chosen.
RNA was extracted with Trizol (Life Technologies), and contaminating
genomic DNA was removed by DNAse treatment (GenHunter Corporation).
cDNA was synthesized with 1 of the oligo dT primers, 40 µmol/L
dNTP, and avian myeloblastosis virus–reverse transcriptase (Roche
Molecular Biochemicals–Boehringer Mannheim) at 37°C. cDNA
was amplified by PCR (94°C, 2 minutes; 40°C, 2 minutes; 72°C, 30
seconds; 40 cycles) with [33P-
] dATP (Dupont
NEN), 2 µmol/L dNTP, 1 random primer (0.5 µmol/L), 1
oligo dT primer (2.5 µmol/L), and Taq DNA
polymerase (Roche Molecular Biochemicals–Boehringer Mannheim).
Amplified products were separated on a 6% denaturing
polyacrylamide gel (Roche Molecular
Biochemicals–Boehringer Mannheim). Bands shared between GCA
temporal artery specimens and not present in control specimens or
in polyclonally activated PBMCs in 2 independent reactions were
eluted and reamplified. PCR products were cloned into the pCR 2.1
vector with the TA cloning kit according to manufacturer's
instructions (Invitrogen). Plasmids grown in transformed INVF' cells
were isolated with the Qiagen plasmid kit (Qiagen). Inserts of the
plasmids were analyzed with the use of automated sequencing.
The GCG program (Genetics Computer Group) was used for homology
searches to match obtained sequences with known genes.
RNAse Protection Assay
Templates for riboprobes were generated by amplifying the
inserts in the pCR 2.1 vector with T7-linked vector universal primers
(T-7 antisense 5'-GAA TCC TAA TAC GAC TCA CTA TAG GGA GGT AAC GGC CGC
CAG TGT GCT G-3'; sense 5'-TAA CGG CCG CCA GTG TGC TG-3'). The
templates for the GAPDH probe were generated by PCR with GAPDH-specific
primers (T-7 antisense 5'-GAA TCC TAA TAC GAC TCA CTA AGG GAG GGG CGT
CTT CAC CAC CAT GG-3'; sense 5'-GGC GTC TTC ACC ACC ATG-3') to retrieve
a 150-bp fragment. Riboprobes were obtained by T7 polymerase in vitro
transcription with Maxiscript (Ambion) in the presence of
[32P-] UTP (Dupont NEN) and were purified by
gel electrophoresis with an 8-mol/L urea denaturing 5%
polyacrylamide gel and subsequent elution in 0.2% SDS, 1
mmol/L EDTA, and 0.5 mol/L ammonium acetate.
The RNAse protection assays were performed with Direct Protect according to manufacturer's instructions (Ambion). All samples were adjusted for GAPDH mRNA content. In brief, specimens were lysed in lysis buffer that contained guanidine thiocyanate and hybridized overnight at 37°C in the presence of molar excess of radioactively labeled riboprobe. Single-stranded RNA was digested with RNAse. mRNA-riboprobe hybrids were alcohol-precipitated, separated on a native 5% polyacrylamide gel, and semiquantified with the Molecular Imager for 32P (Bio-Rad Laboratories). A serial dilution of RNA that contained the appropriate sequence was included in every RNAse protection assay for internal standardization.
Antibodies
Cells with upregulated mitochondrial proteins were identified
with mouse mAb against 65-kDa mitochondrial antigen (1:100; Biogenesis)
and cytochrome oxidase subunit 1(1:100; Molecular Probes). Mouse mAb
against CD3 (1:100), CD68 (1:150; PG-M1 or KP-1), and human -smooth
muscle actin (1:50) as well as all secondary and horseradish
peroxidase–conjugated antibodies were obtained from Dako. Rabbit
polyclonal Ab against MMP-2 (1:2000) was kindly provided by Dr. W.
Stetler-Stevenson, NIH, Washington, DC.13 HNE-modified
amino acid residues (lysine, histidine, and cysteine) on protein were
detected by a rabbit polyclonal Ab (1:1000).14
Streptavidin-conjugated alkaline phosphatase and Vector Red
substrate kit were purchased from Vector Laboratories. Goat anti-rabbit
Ig Cy2-conjugated Ab (1:200) was obtained from Jackson
ImmunoResearch.
Immunohistochemistry
Five-micrometer paraffin sections were dewaxed, and
endogenous peroxidase was blocked. After antigen recovery
with steam, nonspecific binding was blocked with 5% rabbit serum (Life
Technologies). To stain mitochondria, sections were incubated with
mouse mAb against 65-kDa mitochondrial antigen overnight or mouse mAb
against cytochrome oxidase subunit 1 for 30 minutes. Subsequently, the
slides were developed with biotinylated secondary Ab,
streptavidin-coupled alkaline phosphatase, and the Vector Red staining
kit. For 2-color immunohistochemistry, sections were blocked with 5%
normal goat serum (Life Technologies) and incubated with mAb against
CD68 (PG-M1), -smooth muscle actin, CD3, or MMP-2 for 30 minutes and
developed with horseradish peroxidase and 3,3'-diaminobenzidine (Sigma
Chemical) as chromagen. Nuclei were counterstained with hematoxylin.
Negative controls without primary Ab were included in every series.
Single- and double-positive cells were counted with the use of an
Axiophot microscope (Zeiss). For double
immunofluorescence, sections were incubated with Ab
against MMP-2 for 30 minutes and subsequently stained with
fluorescent goat anti-rabbit Ig Cy2-labeled Ab. Nuclei were
counterstained with 4',6-diamidino-2-phenylindole (DAPI;
Sigma).
To stain HNE adducts in proteins, 5-µm frozen sections from temporal artery specimens were fixed in acetone and 1% paraformaldehyde. After the slides were blocked with 5% normal goat serum, they were labeled with rabbit polyclonal Ab against HNE-modified amino acids by overnight incubation at 4°C. Sections were developed with secondary Ab, alkaline phosphatase, and Vector Red. Adjacent sections were stained with mAb against 65-kDa mitochondrial antigen (1:100) in identical fashion.
Computer Imaging
The temporal artery sections stained for HNE and 65-kDa
mitochondrial antigen were scanned with a confocal
immunofluorescent laser-scanning microscope (Axiophot, Zeiss)
and analyzed with the KS400 image analysis system
(Kotron Electronics). The stained area was quantified as the percentage
of the total tissue area and separately measured for the different
layers of the arterial wall. Double
immunofluorescent sections stained for 65-kDa mitochondrial
antigen and MMP-2 were scanned and analyzed for single- and
double-positive areas with the same imaging system.
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Results |
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Upregulation of Mitochondrial-Encoded Genes in Temporal Arteries Affected by GCA
To search for mechanisms that contribute to vascular damage in GCA, DD-PCR was used to identify genes differentially expressed in the inflamed arterial tissue. Temporal artery samples from 5 patients with GCA were compared with specimens from 5 patients without histomorphological evidence of arteritis and without clinical evidence of polymyalgia rheumatica. To exclude genes commonly expressed in activated mononuclear cells, RNA was also isolated from polyclonally stimulated PBMCs. Bands were considered to be differentially expressed if they were reproducibly upregulated in at least 2 independent DD-PCR reactions and if they were found in at least 4 of the 5 patient samples and none of the controls. Within the arbitrarily chosen window of 10 random primers and 4 oligo dT primers, 19 bands were found that fulfilled these criteria. Sequence analysis of the differentially expressed amplification products and a homology search revealed matches with known gene products for 9 sequences; 10 sequences were unknown. Three of the predicted gene products were encoded by mitochondrial DNA. Sequences highly homologous to 12S rRNA, NADH dehydrogenase subunit 4, and cytochrome oxidase subunit 1 were found to be specifically overexpressed in the inflamed arteries compared with noninflamed arteries and in vitro–activated PBMCs. The 2 enzymes, NADH dehydrogenase subunit 4 and cytochrome oxidase subunit 1, belong to complexes I and IV of the respiratory chain, respectively.
To confirm overrepresentation in GCA arterial
tissue, the expression levels of the mitochondrial-encoded gene
transcripts were measured with RNAse protection assays (Figure 1
). Quantification of mRNA levels
revealed a 2-fold upregulation of 12S rRNA in temporal artery specimens
from GCA patients. Cytochrome oxidase subunit 1 expression increased
1.5-fold.
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Mitochondrial Activation Is a Characteristic Feature of CD68+
Macrophages that Reside Along the Elastic Laminae and in
the Media
The mitochondrial origin of 3 gene products identified by
DD-PCR suggested a special role for mitochondrial function in the
inflammatory response of GCA. To identify the cell of origin of
upregulated mitochondrial genes in the vascular lesions, sections from
temporal arteries of patients with GCA were stained with 2
mitochondria-specific antibodies that recognize a 65-kDa mitochondrial
antigen or cytochrome oxidase subunit 1 (Figure 2A and 2C, respectively). Both antibodies
displayed the same differential staining pattern in the
arterial wall. They preferentially stained cells located
around the internal elastic lamina and in the media, which suggests
that this subpopulation of cells had upregulated expression of
mitochondrial proteins (Figures 2 and 3). No immunostaining
with either antibody was found in temporal arteries negative for
arteritis (Figure 2E). The lack of staining with antibodies
specific for mitochondrial antigens in normal arteries and the intimal
and adventitial tissue of inflamed arteries was consistent with
previous reports that indicated that immunocytochemical demonstration
of mitochondrial products is restricted to selected tissues and
cells.15 Immunolocalization of mitochondrial products
in GCA arteries demonstrated a layer-specific expression of
mitochondrial products in the arterial wall and
suggested an altered functional state of a cell population localized in
the media and at the media-intima junction. Two-color
immunohistochemistry with T cell, macrophage, and smooth muscle
cell (SMC) markers revealed that nearly all cells with enhanced
transcription of mitochondrial genes expressed the CD68
phenotype (Figure 3A). Cells characterized by high
levels of mitochondrial antigens included multinucleated giant cells
(Figure 3B). In addition, intense staining was a characteristic
finding for CD68+ macrophages arranged in granulomata. CD3+
lymphocytes with mitochondrial activation were rare (<2%) and few
cells that expressed smooth muscle actin had upregulated transcription
of mitochondrial genes.
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We have previously described that tissue-infiltrating CD68+ cells can
be subdivided into distinct subsets on the basis of their topography
and their functional profile.6 To explore whether
mitochondrial activation was associated with a certain topographical
organization and functional commitment, the frequencies of CD68+ cells
staining with 65-kDa mitochondrial antigen specific antibodies were
determined in the different layers of the arterial wall in
a series of patients (Figure 4).
Upregulation of mitochondrial genes was infrequent among CD68+ cells in
the adventitia (median, 5%) although these macrophages are
known to be activated and produce monokines.6
Also, only 
15% of CD68+ macrophages in the intima were
characterized by increased mitochondrial transcription. In contrast,
between 38% to 68% of macrophages around the internal and
external elastic laminae stained positive for the antibodies. In
summary, upregulation of mitochondrial gene transcription was a typical
finding for a subpopulation of macrophages that infiltrate
tissue and for multinucleated giant cells, which suggested that these
cells had unique functional capabilities.
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Cells With Upregulated Mitochondrial Gene Expression Colocalize
With Membrane Destruction Caused by Oxidizing Free Radicals
The topographical restriction of CD68+ cells with upregulated
mitochondrial transcription to the center of tissue damage in the blood
vessel wall suggested that these cells were specialized to inflict
arterial injury. Possible mechanisms include the release of
ROS and the production of MMP. One of the stable sequelae of
oxidative damage in tissue is lipid peroxidation of fatty acids in
membrane bilayers. ROS readily attack 
-3-polyunsaturated fatty acids
of membrane phospholipids that results in the release of toxic
aliphatic aldehydes such as malondialdehyde bis-(diethyl acetal)
and HNE.12 16 Malondialdehyde and HNE destructively
interact with proteins or nucleotides to form adducts. HNE
protein adducts were readily detectable on multinucleated giant cells
and on macrophages that surrounded the elastic laminae and on
macrophages in the media (Figure 5). Diffuse tissue staining with
HNE-specific antibodies was typical for granulomata in which
mononuclear cells as well as surrounding tissue were strongly positive.
In addition, confluent stains of SMC membranes were found in selected
areas of the media. Lipid peroxidation of cell membranes was rarely
spread throughout the entire medial layer but instead displayed a
patchy distribution.
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To establish whether mitochondrial activation and oxidative injury of
cells coincided, the tissue distribution of both phenomena was
correlated in sections from 5 patients with GCA (Table 2). There
was a close anatomic relationship between CD68+ cells with upregulated
mitochondrial proteins and tissue regions altered by ROS-mediated
peroxidation. Not only were the CD68+ cells with mitochondrial
activation targets for oxidative damage, they were also surrounded by
ROS-damaged cells (Figure 5A
and 5B). The colocalization of both
phenomena strongly suggests that ROS are generated in these specialized
macrophages in which they cause autodamage and are involved in
widespread cytopathic events that target the SMC layer.
Co-occurrence of ROS and MMP-2 Production in CD68+
Macrophages Accumulating in Areas of Tissue
Destruction
We have previously described that a fraction of CD68+ cells
arranged along the elastic laminae and infiltrating the media are
characterized by their ability to produce MMP-2.6 Recent
reports have emphasized that MMPs are produced as proenzymes and that
ROS but not NO have an active role in releasing the functional
enzyme.17 To assess whether MMP-2 and ROS
production could be assigned to identical or distinct cells and
could be correlated with the upregulation of mitochondrial gene
transcription, 2-color immunofluorescence was used
(Figure 6A). Positivity for both markers,
anti-MMP-2 and anti–65-kDa mitochondrial antigen, was frequently
encountered. Multinucleated giant cells were consistently
positive for MMP-2 production and mitochondrial activation.
Sixty percent to 95% of MMP-2+ cells in the media and the adjacent
elastic laminae also stained with anti–65-kDa mitochondrial antigen Ab
(Figure 6B). A small proportion of cells had the ability to
secrete MMP-2 in the absence of increased mitochondrial activity.
Mitochondrial activation without MMP-2 synthesis was distinctly
infrequent. Thus, the characteristic functional profile of
CD68+ cells that reside in the SMC layer and
around the elastic membranes that define the media-adventitia and
media-intima junctions includes the production of proteolytic
enzymes, mitochondrial activation, and the release of ROS. Findings
identify these macrophages as cells with a high potential for
tissue injury.
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Discussion |
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GCA is a systemic vasculitis that causes a spectrum of clinical symptoms. Manifestations of systemic inflammation, such as a brisk, acute-phase response; fever; anemia; and weight loss can dominate the clinical presentation.1 However, the most serious complications arise from inflammation-mediated damage of the extracranial arteries and the aorta. Arterial wall destruction of the aorta that results in aneurysm formation and rupture is a disastrous consequence.18 19 Arterial occlusion can cause blindness and stroke. Although the inflammation appears to be a sequelae of antigen recognition by T cells in the adventitia, the actual mechanisms of tissue injury are unclear. The present study provides strong, albeit indirect, evidence that specialized macrophages and giant cells mediate media destruction in this disease through their ability to release ROS and secrete MMP-2. The highly destructive potential of this defined subset of macrophages and giant cells was associated with the overexpression of mitochondrial products. Because these cells can be molecularly and functionally differentiated from other tissue-infiltrating macrophages and from resident cells, it might be possible to target them specifically.
The release of toxic oxygen products has not been considered as a disease component in GCA. Conversely, the formation of ROS has been accepted as a critical pathway of pathology in other vasculitic syndromes. A role of oxidizing free radicals has been discussed in Wegener's granulomatosis, a vasculitis characterized by the emergence of autoantibodies to enzymes in neutrophils. It is possible that these autoantibodies can recognize their target antigens on the surface of neutrophils and directly induce respiratory burst.20 In contrast to Wegener's granulomatosis, neutrophils are virtually absent from the inflammatory infiltrate in GCA. The histologic hallmark of GCA is granulomas with the accumulation of macrophages, lymphocytes, epithelioid cells, and multinucleated giant cells. This histomorphology is highly suggestive of a central role of macrophages in the arterial injury induced by GCA.
We explored whether oxidative stress and oxygen radical–mediated
tissue damage has a role in GCA. ROS-related pathology was suggested by
the typical granuloma formation in GCA. In experimental
pulmonary granulomatosis, high quantities of superoxide anions
are released synchronously with the development of
granulomata.21 Treatment of experimental animals with an
oxygen scavenger, D--tocopherol,
inhibited granuloma development.21 Also, enzymatic
suppression of oxygen metabolites could prevent granuloma
formation.21 Granulomatous reactions are followed by
significant damage to the underlying tissue structures. Extensive
fibrosis usually accompanies resolution of the granuloma and results in
irreversible disruption of tissue architecture and physiology. To
provide evidence for a direct action of oxygen-derived free radicals in
the arterial wall, we searched for adduct formation on cell
membranes. One of the toxic mechanisms of ROS is lipid peroxidation.
HNE production and subsequent formation of Michael adducts with
amino acids is a marker of lipid peroxidation.12 HNE was
detected through the use of specific antibodies that recognize these
adducts on the amino acids lysine, histidine, and cytosine.
This approach has been shown to be a highly sensitive tool to measure
the action of toxic ROS in vivo.14 The staining pattern
obtained by the anti-HNE Ab demonstrated peroxidized lipids on the
surface of CD68+ cells and also on surrounding CD68- cells. Preferred
targets for lipid peroxidation were SMCs in the media. ROS-induced
damage to cell membranes was absent in the intimal and adventitial
layers, which emphasized that direct tissue injury focuses on the media
and the bordering elastic lamina. The congruent distribution of cells
with mitochondrial activation and ROS-induced tissue damage indicated a
causal relationship between these 2 phenomena. The respiratory burst of
phagocytes is usually mediated by NADPH oxidase translocated to the
cell membrane, and cells may need to be metabolically
hyperactive to sustain this activity.22 However, it has
been reported that this enzyme is downregulated as monocytes
differentiate;23 thus, this pathway may no longer be
dominant in the tissue lesions. Alternatively, it could be hypothesized
that mitochondrial activation is directly involved. Increased enzymatic
reactions of the electron transport chain in mitochondria are
associated with a leakage of ROS that may be sufficient to induce lipid
peroxidation.
The generation of ROS in the arterial wall may not only inflict direct tissue damage but may also be involved in the regulation of the activity of proteolytic enzymes. We have previously reported that a specialized population of CD68+ cells synthesizes MMP-2.6 MMP-2+ CD68+ cells can be defined by their localization between the medial SMC and in close vicinity to the internal and external elastic laminae. MMPs are synthesized as proenzymes and need further processing to release active enzymes.9 A recent study by Rajagopolan et al17 has linked the action of ROS with the regulation of activation of MMPs in foam cells isolated from atherosclerotic plaques. To investigate the relationship between the formation of oxidants and the synthesis of MMP-2, we examined the co-occurrence of these 2 macrophage functions in the vascular lesions. Mitochondrial activation overlapped closely with MMP-2 formation and indicated that the production of ROS and MMP-2 identifies a defined macrophage subset. A unique subset of this macrophage population is multinucleated giant cells. These macrophages, as well as the giant cells, reside in the center of tissue injury and appear to be cell populations with the potential to be highly destructive.
The present study has raised several questions that must still be
studied. Curiously, upregulation of mitochondrial proteins was not a
feature of all tissue-infiltrating cells, although that would have been
expected. Also, medial SMCs were not among the cells that overexpressed
cytochrome oxidase or 65-kDa mitochondrial antigen. The
phenotype of increased production of mitochondrial
products was strictly limited to a subset of cells: CD68+
macrophages and giant cells in a defined topographical
localization. It would be most interesting to explore the specific
signals that induce MMP production and upregulation of
mitochondrial transcription with subsequent increase of mitochondrial
activity. Previous studies have shown that IFN- produced by T cells
in the adventitia is the key cytokine of the inflammatory
response in GCA.5 IFN- is known to be a potent
macrophage activator; however, additional stimuli
may determine the activation of these medial macrophages and
their pattern of gene expression. More importantly, macrophage
differentiation and activation appears to vary among GCA patients, and
these differences correlate with the extent of arterial
wall injury and remodeling.24 GCA patients with minimal
intimal hyperplasia have a largely preserved arterial
architecture with minimal destruction of the elastic lamina, whereas
patients with marked intimal hyperplasia are characterized by a
disrupted internal elastic lamina. These histologic differences do not
appear to be different stages of the disease process but instead
reflect heterogeneity in macrophage function.
Medial macrophages in patients with intimal hyperplasia have a
different product profile and tend to form giant cells more
frequently compared with patients without intimal
proliferation25 (M.K., J.J.G., C.M.W., unpublished data,
1998).
The understanding of disease mechanisms operative in the damage to the
arterial walls in GCA has obvious implications. Molecules
implicated in causing tissue injury are prime candidates for
therapeutic inhibition. Currently, there is only 1 treatment that has
been shown to be efficacious: the use of
corticosteroids given in large doses over several
years. This treatment is complicated by a high rate of serious side
effects that must be weighed against the risk for blindness, stroke,
and aneurysm development. Constraints in the therapeutic
options reflect the paucity of information on mechanisms operative in
arterial injury. If proteolytic destruction by selected
MMPs constitutes an important attack on the medial layer, therapeutic
inhibition of proteases should be considered. Approaches could also be
directed toward the inhibition of ROS. The tissue-damaging effects of
toxic ROS may be reduced through the use of oxygen scavengers such as
D--tocopherol. Because some of these
approaches are rather nontoxic, direct application in clinical trials
could be considered. Other interventions that are of a more
experimental nature should be tested in a model system. We have
recently succeeded in establishing a novel "animal model" for GCA.
Granulomatous arteritis can be maintained in temporal artery specimens
collected from patients and engrafted into severe combined
immunodeficiency mice.26 We have used these artery-mouse
chimeras to investigate the mechanism of action of
corticosteroids in vivo.27 Therapeutic
interventions directed at tissue injurious pathways could be rapidly
explored in the engrafted arteries. Finally, it is likely that
tissue-injurious reactions in the temporal artery of patients with GCA
are not unique to vasculitis. It can be predicted that the mechanisms
of arterial injury are shared in a variety of inflammatory
and noninflammatory diseases that affect muscular arteries. The
spectrum of diseases may include atherosclerotic disease that
affects coronary arteries that have many structural features in
common with temporal arteries. Understanding the disease mechanisms in
GCA may encourage the search for therapeutic interventions in
atherosclerotic disease.
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Acknowledgments |
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This work was supported in part by the Mayo Foundation and a grant from the National Institutes of Health (NIH; RO1 EY11916) and the Research to Prevent Blindness Inc (RPB No. 3). Drs Rittner, Kaiser, and Brack were supported by fellowships from the Deutsche Forschungsgemeinschaft. We appreciate the kind gift of anti-MMP-2 antibody from Dr W. Stetler-Stevenson, NIH, Washington, DC. We thank Dr Johannes Björnsson from the Department of Pathology and our colleagues from the Department of Ophthalmology, Mayo Clinic, for their support and the patients who donated the tissue to make these studies possible. We also thank Toni L. Higgins for secretarial assistance.
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Footnotes |
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Reprint requests to Dr Cornelia Weyand, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
Received August 10, 1998; accepted February 24, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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