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Regular Articles |
From the Department of Laboratory Medicine,*
Vaccine/Virology Division, Retrovirology Laboratory, Seattle,
University of Washington School of Medicine, Washington; the Department
of Laboratory Medicine,
Yale University, New
Haven, Connecticut; and the Department of Pulmonary and Mediastinal
Pathology,
Armed Forces Institute of
Pathology, Washington, D.C.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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80%.
Thus, infiltration of alveolar septae with CD8+ T
cells was highly correlative with VCAM-1/VLA-4 adhesive
interactions, and focal expansion of B cells was coincidental
to co-infection with EBV.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aggressive B-cell non-Hodgkin lymphomas and fatal polyclonal lymphoproliferative disorders occur with increased frequency among children with HIV AIDS.5,8-11 Many of these neoplastic and pre-neoplastic conditions have been attributed to co-infection with human herpesviruses.12-15 Interactions between herpesviruses and HIV have been demonstrated in vitro through experiments showing transactivation, CD4 up-regulation, Fc receptor induction, pseudotype formation, cytokine production, and antigen presentation.16-18 Still, the role of human herpesviruses in LIP, strictly as opportunists or as co-factors in HIV disease, is yet unknown.
Cell-to-cell and cell-to-extracellular matrix interactions are
important in cell migration. The binding of lymphocytes to vascular
cell adhesion molecule-1 (VCAM-1), E-selectin, and/or intracellular
adhesion molecule-1 (ICAM-1) expressed on vascular endothelial cells
accounts for a significant portion of the binding of lymphocytes to
endothelium.19-22
Collectively, these molecules differ in
their leukocyte binding repertoire. The integrin 
4ß1 that defines
very late activation antigen-4 (VLA-4), is the leukocyte ligand for
VCAM-1 and is expressed by a restricted set of cell types, including
monocytes21,23
and lymphocytes,22,24,25
and
is present at low levels on some polymorphonuclear
cells.26,27
E-selectin also mediates the adhesion of
polymorphonuclear cells,28
monocytes,29
and
certain lymphocyte subsets.19,30
In contrast, ICAM-1
promotes the adhesion of all leukocytes that bear the surface receptor
leukocyte function-associated antigen-1 (LFA-1).31,32
The release of chemotaxins in concert with the expression and avidity of specific adhesion molecules are mechanisms that influence inflammatory cell migration and localization of cells to specific tissue sites, as blocking of adhesion molecule receptors in vivo can influence subsequent cellular infiltration.21,22,33 Inasmuch as cell trafficking and extravasation of lymphocytes to the lung may be controlled by the differential expression of these endothelial and/or leukocyte adhesion molecules, dysregulation of these processes can lead to immunopathological disease. Herein, we show that pulmonary infiltration by CD8+ T lymphocytes was highly associative with VCAM-1/VLA-4 adhesive interactions and, although HIV infection and AIDS were requisite for the condition of LIP, co-infection with Epstein-Barr virus (EBV) significantly compounded the severity and character of disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lung was obtained at autopsy or by open-chest biopsy from 11
prepubescent children (median age, 3.6 years) with perinatally acquired
HIV-1 infection and classified as P2-C (symptomatic) according to the
CDC Control and Prevention classification system. All subjects had
radiological evidence of pulmonary interstitial reticulonodular
infiltration (widespread subsegmental consolidations) in the absence of
pulmonary opportunistic infections, including Pneumocystis
carinii, or neoplasia by standard histopathological, cytological,
and/or microbiological culture and staining techniques. Exceptions were
patients with human herpesvirus infections, where virus was detected
prospectively in lung by in situ hybridization. Of these 11
cases, 6 had histological evidence of moderate to severe LIP, and of
these 6, 3 showed vascular lesions manifest by thickening of the
arterial intimae (Table 1)
. The
microscopic features of the vascular lesions and their association with
HIV and other infectious agents were not known before the initiation of
the study. Samples from the remaining five children were diagnosed with
mild nonspecific interstitial pneumonitis, and none had pulmonary
vascular lesions. In addition, postmortem lung was obtained from
children without HIV infection or pneumonia (n =
4) and from children with AIDS but without LIP or pulmonary
arteriopathy (n = 2). For in situ
hybridization and most immunohistochemical analyses, representative
sections of lung were fixed in 10% neutral buffered formalin, embedded
in paraffin wax, sectioned to a thickness of 5 µm, and mounted on
silane-treated (triethoxyaminopropyl-silane, American HistoLabs,
Gaithersburg, MD) glass microscope slides. Lung tissue was also
snap-frozen in 2-methylbutane and embedded in OCT compound (Miles,
Elkhart, IN) for use with antibodies that precluded aldehyde fixation.
Serial sections were cut on a cryostat microtome at a thickness of 7
µm for immunohistochemical analyses and 10 µm for tissue adhesion
assays and allowed to air dry at room temperature for 2 hours.
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Tissues were evaluated by hematoxylin and eosin (H&E) stains for patterns of leukocyte infiltration consistent with LIP. In addition, special stains, including Masson's trichrome for collagen, Weigert's resorcin fuchsin for elastic fibers, Von Kossa's for mineral, Congo red for amyloid, and phosphotungstic acid hematoxylin for fibrin, were used to characterize the composition of vascular lesions in paraffin-embedded tissues. When available, frozen sections were also evaluated for lipid by staining with oil red o.
Immunohistochemical procedures were used to 1) identify cell types
within inflammatory and/or arteriosclerotic lesions, 2) identify cells
expressing viral antigens, and 3) determine the level of expression of
endothelial and leukocyte adhesion molecules in histological sections
of lung from individuals identified in Table 1
. Immunophenotyping was
performed following standard procedure,34,35
which
included an antigen retrieval step when necessary (steaming in 1 mmol/L
citrate buffer, pH 6.0, for 10 to 20 minutes) and used monoclonal
antibodies (MAbs; 10 to 20 µg/ml; MAb clones and sources are
identified in figure legends) to viral and cellular antigens. Briefly,
primary MAbs were applied overnight at 4°C followed by detection with
isotype-specific secondary antibody and an ABC peroxidase technique
using 3,3'-diaminobenzidine (Vector Laboratories, Burlingame, CA) as
the chromogen. To control for nonspecific binding, isotype-matched
antibodies for irrelevant antigens were used in substitution of the
primary antibody. Stained tissues were evaluated by incident light
microscopy.
Tissue Adhesion Assay
Leukocyte adhesion to vascular endothelium was assessed by a
modified tissue adhesion assay first described by Stamper and
Woodruff36
and used more recently by Sasseville et
al.21
Briefly, frozen sections were pretreated with RPMI
1640 containing 10% fetal bovine serum for 10 minutes at 4°C. Next,
106
CD20+ B cells (human B cell line Ramos) or
106
CD8+ T cells (primary clones LN2A3,
LN33D7–797, and LN9E12, as described previously37
) were
diluted in 100 µl of medium and applied to each tissue section, and
the section and cells were gently shaken for 30 minutes at 4°C. After
mitogen stimulation for 48 hours (5 µg/ml pokeweed mitogen for B
cells and 5 µg/ml phytohemagglutinin for T cells; Sigma Chemical Co.,
St. Louis, MO), the clones were shown to express both VLA-4 4 and
ß1 subunits when assessed by immunocytochemistry (Figure 1P
, inset) and by flow cytometry. In
addition, unstimulated BCBL-2 cells, a B cell lymphoma-derived cell
line,38,39
did not express VLA-44ß1 and
were therefore used to control for nonspecific adhesion. The sections
were then gently rinsed in cold 0.15 mol/l PBS, fixed in 1%
glutaraldehyde in PBS for 15 minutes at 4°C, rinsed in PBS/0.2%
gelatin (pH 7.2), and subsequently stained with 0.5% toluidine
blue/30% ethanol for 15 to 20 seconds. After rinsing in 100% ethanol,
the stained sections were mounted and examined microscopically.
Experiments were performed a minimum of five times.
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4ß1 on CD8+ T cell lines. Still, a
similar approach was used to assess the potential role of other
adhesion pathways in LIP, including ICAM-1/LFA-11ß2
and E-selectin. Briefly, before the tissue-cell incubations, tissue
sections, cells, or both were incubated for 30 minutes at 4°C with
saturating concentrations (20 to 40 µg/ml) of MAbs to VCAM-1 (clone
51–10C9, PharMingen, San Diego, CA) or the 4ß1 subunits of VLA-4
(clones HP2/1 and Lia1/2, respectively, Coulter-Immunotech, Westbrook,
ME). Monoclonal antibodies to CD31 (pan-endothelial (clone JC/70A),
DAKO, Carpinteria, CA), and CD45 (leukocyte common antigen, (clone
HI30), PharMingen, San Diego, CA) were used as controls on tissues
and cell lines, respectively. In Situ Hybridization
HIV-1 RNA probes were synthesized with 125I-labeled CTP (Amersham Corp., Arlington heights, IL). Five sense and five antisense probes, in all representing 90% of the HIV-1 genome, were synthesized using pGem 3 subcloned restriction fragments of the clone HXB2, as described previously.40 The probes were then applied to tissues as a cocktail. For EBV, 30-bp antisense oligonucleotide probes, one complementary to the EBER-1 gene transcript and another to the BHLF-1 gene transcript were prepared by labeling the 3' end with digoxigenin-11-dUTP (DIG) using a DIG-tailing reaction (Genius 6 kit, Boehringer Mannheim, Indianapolis, IN). The probes were applied to tissues as described previously.41 The EBER-1 gene is actively transcribed in latently infected cells, and the BHLF-1 gene is expressed early in the EBV life cycle and therefore identifies EBV-infected cells in the replicative phase of the virus life cycle. In addition, DIG-labeled oligonucleotide probes specific for the cytomegalovirus (CMV) early gene transcript were used following manufacturer recommendations (Novocastra, Newcastle, UK) and as reported previously.42 Expression of the CMV early gene transcript precedes DNA replication, and therefore, the probe allows for the earliest detection of permissive infection. Last, DIG-labeled RNA probes complementary to the Kaposis sarcoma herpesvirus (KSHV) T0.7 transcript (155 bp) were constructed as described previously.38,39 The T0.7 gene product is transcribed at a high molar ratio during viral latency. Positive and negative controls consisted of cytocentrifuge preparations of primary peripheral blood mononuclear cells (PBMCs) and/or lung tissues known to be positive or negative for the respective virus by DNA extraction followed by solution-based polymerase chain reaction (PCR) and liquid hybridization. Hybridization controls consisted of noncomplementary (sense-strand) 125I-labeled RNA probes for HIV and noncomplementary DIG-labeled probes for KSHV, EBV, and CMV of similar length and G/C content as the antisense probe. After development, the slides were examined by incident light and/or dark-field microscopy.
Semiquantitative Analysis
LIP was defined as infiltration of the pulmonary interstitium with
mostly CD8+ T lymphocytes and in some individuals by
expansion of the alveolar septae with dense aggregates of polymorphic
and polyclonal (CD19+ and CD20+) B lymphocytes.
The severity of LIP was scored using a system we described
previously35,43
: +, multifocal interstitial
leukocytes; ++, multifocal and intermittent confluent areas of
leukocyte infiltration; and +++, confluent areas of alveolar septal
thickening with leukocytes and multifocal areas of lymphocyte
aggregation (Table 1)
.
The degree of expression of endothelial VCAM-1, ICAM-1, and E-selectin was assessed semiquantitatively using a method modified from that previously described.44 Briefly, both numbers of immunoreactive pulmonary vessels in a 0.5-cm2 sample and intensity of staining were used as criteria. For relative numbers of immunoreactive vessels, assigned scores included the following: 0, no vessels; 1, 1 to 4 vessels; 2, 5 to 10 vessels; 3, 11 to 15 vessels; 4, 16 to 20 vessels; and 5, >20 vessels. The scores for staining intensity were as follows: 0, no reactivity; 1, faint; 2, moderate (±4 vessels being intense); 3, intense staining of >4 pulmonary vessels. The sum of these two values for numbers and intensity of immunoreactive vessels were then used as the final score, which had a theoretical range from 0 to 8. Each sample was evaluated independently and in a blind fashion by two reviewers (S.J. Brodie and K. Diem). The severity of LIP, extent of macrophage viral burden, and levels of vessel immunoreactivity were compared using a Spearman nonparametric correlation analysis where r values >0.7 and P > 0.05 were considered significant.
Cell adhesion to tissue sections was assessed by computerized image
analysis. Images from 10 representative 10x microscopic fields were
transmitted to a computer equipped with a digital imaging board and
software for determination of point count size from which a percentage
of specifically stained cells per linear unit (0.2 mm) of endothelium
was then determined. We chose 0.2 mm as the unit of measurement. This
allowed for the inclusion of most intralesional pulmonary venules.
Next, a binding coefficient was determined by the quotient of bound
cells/vascular dimension unit.21
Cells that bound to
venular endothelium were defined as those adhering exclusively to the
luminal (apical) endothelial surface and were differentiated from
resident or inflammatory leukocytes by concurrent assessment of
untreated consecutively sectioned tissues. At least 10 vessels 0.2 mm
in circumference from each tissue section were used to obtain a mean
binding coefficient (MBC). Data were analyzed by one-way analysis of
variance (ANOVA) to test whether the mean differed among groups of
individuals categorized by HIV status and histopathology (Table 2)
. A Bonferroni multiple comparison test
was then used as a post-test to compare pairs of group means where
P > 0.05 was considered significant. Inhibition of
cell adhesion was measured as the percentage of MBC obtained using
blocking or control antibodies relative to the MBC obtained from a
serial tissue section in the same assay without antibody treatment. To
determine whether cell binding occurred on endothelium expressing
VCAM-1, ICAM-1, or E-selectin, after performing the adhesion assay,
tissue sections were post-fixed in 0.5% glutaraldehyde. An
immunoperoxidase technique was then performed on the same tissue
section using antibodies to VCAM-1 (clone 51–10C9, PharMingen, San
Diego, CA), ICAM-1 (clone HA58 (CD54), PharMingen) or E-selectin (clone
68-5H11 (CD62E), PharMingen), respectively. Immediately after chromogen
development, the tissue sections were post-fixed in 2%
paraformaldehyde/0.5% PBS for 10 minutes at 25°C, washed in
PBS/0.2% gelatin, stained for 15 to 20 seconds in toluidine blue/30%
ethanol, rinsed two times in 100% ethanol, and mounted.
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Results |
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The six subjects with LIP (Table 1)
all showed widespread
hyperplasia of bronchus-associated lymphoid tissue (BALT) and
lymphocyte infiltration of alveolar septae (Figures 1A and 3A)
. BALT,
defined as resident organized lymphoid tissue of the lung, was observed
only in a small percentage (one of six) of children without evident
pulmonary infection. This finding is consistent with other studies
where BALT was present in human fetal and infant lung only when there
was evidence of antigen stimulation.45
Lymphocytes of
CD8+ T cell lineage represented the predominant intraseptal
infiltrate (Figure 1B)
. Three of these six individuals also showed
dense interstitial perivascular aggregates of polymorphic B lymphocytes
(Figure 1C)
, most in replicative stages of the cell cycle (Figure 1D)
and all surrounding well developed networks of follicular dendritic
cells (FDCs; Figure 1, E and F
(double arrow)). These aggregates of B
cells were predominantly immunoblasts and showed normal to slightly
elevated levels of mitotic activity (two to four mitotic figures per
100 cells). In addition, 20% to 30% of these B cells contained EBV
EBER-1 gene transcripts (Figure 1G)
and/or EBV latent membrane proteins
(Figure 1H)
. Herpesviral gene products localized to B cells (Figure 1G
,
inset) and FDCs (Figure 1I)
and not to other cell types, including T
cells (Figure 1J)
. HIV mRNA (Figure 1K)
and protein antigens also
localized within follicular germinal centers (Figure 1K)
in association
with FDCs. Loosely scattered cells within the thickened pulmonary
interstitium and pockets of cells on the periphery of lymphocyte
aggregates contained high copy numbers of HIV gag proteins
(Figure 1L)
, areas shown previously to harbor alveolar and interstitial
macrophages (Figure 1F)
. By combining immunohistochemistry for
cell-surface antigens with in situ hybridization for HIV
mRNA, cells that contained high viral copy numbers were mostly FDCs in
germinal centers (Figure 1K
, inset) and macrophages within interseptal
spaces.
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Pulmonary arteritis and/or thickening of the arterial intimae was
strongly correlative with severe LIP (Table 1)
. Lesions were present in
mostly medium-sized arteries and included components of inflammation,
intimal fibrosis, fragmentation of elastic tissue, and/or fibrosis or
calcification of the media. In the most severely affected vessels, the
endothelium was focally detached from the underlying intimae and the
cells were slightly rounded. Vessels demonstrating intimal thickening
showed variable degrees of luminal narrowing (Figure 2A)
with fragmentation and/or duplication
of the internal elastic lamina (Figure 2B)
. The thickened intimae
consisted of mostly collagen (Figure 2C)
, smooth muscle (Figure 2D)
,
and occasional macrophages, some harboring the HIV genome (Figure 2E)
.
In addition, one patient (case 6) showed extensive mineral deposits
that localized mostly within the adventitia of medium-sized arteries
(Figure 2F)
. Other special stains revealed the absence of amyloid,
lipid, and fibrin within the vascular wall. Platelet and fibrin thrombi
could be visualized within intimal plaques and within the lumen of
smaller arteries. Fibrinoid necrosis, a feature of pulmonary
infarction, was not present in any of the samples examined, nor did any
of the subjects show lesions suggestive of pulmonary hypertension
and/or congestive heart failure, such as widespread pulmonary edema.
Tissues from children diagnosed with nonspecific interstitial
pneumonitis, a common sequelae to HIV infection in
adults,46
and children with AIDS but without lung
involvement had no evidence of vascular disease. Collectively, these
findings suggest an association between pediatric LIP and arteriopathy
in children with HIV-induced immune suppression.
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Three common and distinct pathways mediating mononuclear cell
adhesion to pulmonary endothelium were investigated. VCAM-1 expression
was significantly heightened in vascular endothelium in children with
LIP and was most pronounced in veins with perivascular inflammatory
foci (Figures 1O and 3A)
and included all
tissues with arteriosclerotic lesions (Table 1)
. Interestingly,
arterial endothelium from vessels with thickened intimae expressed low
or undetectable levels of VCAM-1, and there was no evidence of cells
with HIV gag mRNA or p24 antigen expression within the
vascular wall. When ranking the intensity of VCAM-1 expression with a
range in score from 0 to 8, the mean score for children with LIP was
5.5 ± 1.0. Tissues with severe LIP, characterized by
EBV-associated lymphoproliferation and arteriopathy, showed the most
extensive and intense VCAM-1 staining (7.3 ± 0.7). In contrast,
HIV-infected children without LIP, including those with nonspecific
interstitial pneumonitis (1.6 ± 0.5) and those without pulmonary
lesions (0.9 ± 0.4) showed low levels of VCAM-1 immunoreactivity
and were no different from that of HIV-seronegative children without
pulmonary lesions (0.7 ± 0.2; P > 0.05, ANOVA).
Thus, only in tissues with LIP was there a significant difference in
VCAM-1 expression over background (P < 0.05,
ANOVA). VLA-4, the leukocyte ligand for VCAM-1, was expressed at high
density on perivascular lymphocytes (Figure 3B)
, most of which also
bore the CD8 surface receptor.
In contrast with VCAM-1, widespread and uniform expression of endothelial ICAM-1 was observed in sections of lung from all 11 test subjects, as well as samples from the 2 HIV-positive and 4 HIV-negative patients without pulmonary lesions (not shown). Variations in staining intensity were not appreciable. Interestingly, ICAM-1 was also expressed on smooth muscle cells within the intimae of affected vessels. In turn, E-selectin was only weakly and sporadically expressed on endothelial surfaces (not shown) and mostly from patients with nonspecific pneumonitis, many of which also showed chronic-active inflammation.
Using a tissue adhesion assay, we show that CD8+ T cell
clones selectively adhere to endothelium in lung from children with LIP
(Figure 1P)
. The MBC for CD8+ T cells
(VLA-44ß1 positive) was significantly greater when
comparing individuals with LIP (22.2 ± 1.9) with those with
nonspecific pneumonitis (2.3 ± 0.7; P = 0.02,
ANOVA) or to individuals without pulmonary lesions (0.7 ± 0.2;
P < 0.001, ANOVA; Table 2
). There was no difference
when comparing individuals with moderate (20.5 ± 2.6;
n = 3) and severe (23.8 ± 3.1; n
= 3) LIP (P = 0.4, ANOVA). Similar observations
were made between the same groups when comparing tissues for Ramos cell
(B cell) adhesion, although the average MBCs were much lower than those
of CD8+ T cell clones (Table 2)
. This decrease in binding
efficiency of B cells coincided with a marked reduction in the
expression of VLA-44ß1. In all, tissues with a high
ratio of immunoreactive vessels and elevated staining intensity for
VCAM-1 demonstrated the most severe lesions of LIP and conferred the
greatest adhesive interactions to cells expressing the VLA-4 ligand.
Pretreatment of CD8+ T cell clones with blocking
antibodies to VLA-44ß1 (clones HP2/1 and Lia1/2
(CDw49d/CD29), Coulter-Immunotechnology, Westbrook, ME) and/or
pretreatment of tissues with LIP using blocking antibodies to
VCAM-1 (clone 51–10C9 (CD106), PharMingen) resulted in >80%
inhibition of this adhesion (Figure 1Q)
. For example, children with LIP
had an average MBC of 22.2 ± 1.9 for CD8+ T cell
clones per linear unit (0.2 mm) of pulmonary venular endothelium (Table 2)
. When these tissues and cells were pretreated with MAbs to VCAM-1 or
VLA-44/ß1, respectively, the MBC dropped to <5. In
turn, Ramos cells, which also expressed VLA-44/ß1 but
at a much lower intensity than CD8+ T cell clones, also
bound less efficiently to endothelium in serial sections of the same
tissues. Unstimulated cells bound less efficiently than
mitogen-stimulated cells, and unstimulated BCBL-2 cells did not adhere
to vascular endothelium under any application. LFA-1 was also expressed
on CD8+ T cell clones and Ramos cells. However, in the
absence of VCAM-1 expression, neither cell type bound appreciably to
endothelium that expressed ICAM-1 alone (T cells, MBC = 2.4
± 0.8; Ramos cells, MBC = 0.8 ± 0.2; clone HA58 (CD54),
PharMingen) or vessels that co-expressed ICAM-1 and E-selectin (T
cells, MBC = 1.5 ± 0.6; Ramos cells, MBC = 0.7 ±
0.2). Similarly, pretreatment of lymphocyte cell lines with saturating
concentrations of antibodies to LFA-11ß2 (clone HI111
(CD11a), PharMingen, and clone MHM23 (CD18), DAKO) did not
significantly affect the binding of these cells when applied to tissues
with vessels that expressed ICAM-1 or E-selectin alone (T cells,
MBC = 2.1 ± 0.7; Ramos cells, 0.8 ± 0.1;
P > 0.05). Cells and tissues when incubated with
antibodies to CD45 and CD31, respectively, as a control for nonspecific
blocking of cell-to-tissue adhesion, did not alter the MBC in the
tissue adhesion model (P > 0.05).
Virus Localization by In Situ Hybridization
Mononuclear cells bearing HIV transcripts were observed, although
infrequently, within the wall of arteries (Figure 2E)
and veins (Figure 3C)
and mostly in sites of perivascular infiltration. The majority of
cells with detectable HIV mRNA had morphological and phenotypic
(CD68+) features of macrophages and localized within the
interstitial and alveolar spaces (Figure 1, K and L)
. The number of
HIV-infected intralesional monocyte/macrophages was strongly
correlative with the severity of LIP (r =
-0.86; P = 0.01, Spearman rank correlation
coefficient, two-tailed analysis); severe lesions with both
infiltrative and proliferative components (cases 1, 2, and 6) showed
the greatest number of cells with detectable HIV gag gene
expression (Table 1)
.
Because 
-herpesviruses have also been linked to LIP and more
recently with arteriopathy,47,48
we examined lung for the
presence of viral transcripts and protein antigens indicative of CMV,
KSHV, and/or EBV infections. We also examined the effects of concurrent
herpesvirus infection on adhesion molecule expression. The child with
CMV pneumonitis (case 4) had no evidence of arteriopathy and showed
only low levels of endothelial VCAM-1 (score 2.0). Although, VCAM-1
expression in this patient was most pronounced in vessels within areas
of heavy lymphocyte infiltration, the distribution of cells expressing
CMV RNA was widespread (Figure 3E)
and included histologically normal
areas of the lung. The role of KSHV in lesion development was also
unclear as cells containing the T0.7 transcript were detected in only 1
of 11 subjects (case 1), and there were no patterns of virus
localization relative to sites of lymphoproliferation, arteriosclerotic
vessels, and/or vessels expressing VCAM-1. In contrast, the three
patients with severe LIP (Table 1)
characterized by dense intraseptal
aggregates of lymphoblasts (Figure 1C)
, showed that as many as 20% of
these cells harbored detectable levels of EBV EBER-1 RNA (Figure 1G)
and/or expressed EBV latent membrane protein 1 (Figure 1H)
. EBV was not
detected in bronchiolar or alveolar epithelium, as reported by
others,49
nor was it observed in T cells (Figure 1J)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 and ß1
subunits of VLA-4 were applied concurrently. Collectively, these
results suggest strongly that VCAM-1/VLA-4 adhesive interactions are
important in lymphocyte trafficking in LIP. Moreover, differences in
host factors in children that allow for higher viral
loads50,51
and heightened cytokine
responsiveness52
may ultimately influence the progression
of LIP.
ICAM-1 mediates an LFA-1-dependent pathway31
and, like
VCAM-1, may be important in lymphocyte emigration to sites of
inflammation or immune reaction.23,53
However, unlike
VCAM-1, ICAM-1 is constitutively expressed and is present on a variety
of other cell types. Hence, the role of ICAM-1 in inflammation may be
difficult to assertain. Moreover, because ICAM-1 was also expressed in
normal areas of diseased tissue, it was unlikely to have played a major
role in leukocyte infiltration in children with LIP. E-selectin may
also play a role in the initial interaction of certain subpopulations
of T lymphocytes with activated endothelium.19,30
We show
that E-selectin was expressed on venular endothelium from tissues with
nonspecific pneumonitis, most often when there was a prominent
neutrophil component, but was absent or only weakly expressed in
tissues with LIP. Blockade of E-selectin and
ICAM-1/LFA-11ß2 did not significantly alter lymphocyte
attachment to pulmonary endothelium. Thus, ICAM-1 and E-selectin did
not appear to contribute substantially to lymphocyte trafficking in
LIP. Still, we cannot discount that these and other adhesion pathways,
although having a minor contribution in the adhesion assay when
examined individually, may work in concert in vivo to
contribute to lymphocyte-endothelial adhesion. It is also conceivable
that our assays were insensitive to reveal relatively low-affinity
interactions that may occur with selectins and their ligands.
A variety of herpesviruses have also been implicated in pediatric LIP5,8,12,13 and expansion of BALT.54 We show that EBV localized to B lymphoblasts in all tissues with lymphoid aggregates and also within FDCs in lesions with germinal center formation. This high incidence of EBV infection and virus replication may result from defective regulation of EBV in patients with AIDS or AIDS-related disorders.55 Patients with LIP typically show high numbers of EBV-infected B cells in circulation as a consequence of profound defects in T cell immunity.55,56 Children with AIDS or AIDS-related disorders, including those with LIP, are also predisposed to EBV-associated non-Hodgkin's lymphoma.14,15,56 Consequently, it has been proposed that LIP may represent an intermediate process between benign and malignant transformation.57 Co-infection of B cells with HIV and EBV has also been described as a possible co-factor in the progression from polyclonal B cell proliferation to lymphoma as both the up-regulation of c-myc and activation of EBV can occur as a result of HIV infection.11 Interestingly, when comparing the intensity and avidity of VCAM-1 expression and cell adhesion (MBCs) in children with LIP there was no difference when examining tissues with EBV-associated lymphoblastic lesions (+++LIP) and those without (++LIP). This further suggests that this B cell component of LIP does not occur as the result of infiltration, but results from local expansion of B cells.
The pulmonary arterio-occlusive lesions that we describe appear to be
unique to children with AIDS and, interestingly, were observed only in
children with concurrent EBV infection. Similar lesions have been
described in macaques experimentally infected with
SIVmac58
and more recently in transgenic mice
carrying a replication-defective HIV-1 provirus.59
Newly
described -herpesviruses have also been linked to arteriopathy in
SIV-infected macaques48
and large-vessel arteritis in
mice.47
Various types of synergy have been described
between retroviruses and herpesviruses.16-18
Of potential
relevance to vascular disease, HIV-1 tat protein was shown
to promote the growth of normal vascular cells and spindle cells
derived from AIDS-associated Kaposi's sarcoma,60
a tumor
of vascular origin and common neoplasm in AIDS patients and a disease
recently attributed to -herpesvirus infection.61
Studies of Kaposi's sarcoma cell lines indicate that KS cells release
a variety of cellular growth factors.62
EBV-infected
and/or EBV-activated lymphocytes also liberate angiogenic cytokines,
including endothelial growth factor and fibroblast growth
factor.63,64
Thus, we examined sections of lung with
histological evidence of arteriopathy for the -herpesviruses KSHV
and EBV by in situ hybridization. Vascular lesions were
characterized by intimal thickening resulting from collagen and smooth
muscle deposition and affected mostly medium-sized arteries. Only 1 of
11 subjects demonstrated cells carrying the KSHV latent genome, and
unlike EBV where high levels of virus localized to B cells within
lymphoid aggregates, there was no pattern to the distribution of KHSV
in this patient, and viral transcripts were not detected in individuals
with vasculopathy. Previous studies of ours have shown KSHV latent gene
transcripts in prostate glandular epithelium of HIV-infected men but
only at very low viral copy number.38,39
In contrast, both
latent and transcriptionally active EBV infection of B cells occurred
in all individuals with arterio-occlusive disease. Yet, unlike HIV, EBV
was not detected within the vascular wall of affected vessels. Others
have shown the absence of EBV in large-vessel disease.65
We also examined lung for CMV, a ß-herpesvirus capable of
up-regulating HIV replication in macrophages16,66
and
thought to induce smooth muscle proliferation in inflammatory
aortic aneurysms.67
Cytomegaloid cells carrying the CMV
early gene were identified in the pulmonary interstitium but only in a
single patient and not in association with vascular lesions or within
sites of lymphoid aggregation. Collectively, these findings suggest
that EBV, and not KSHV or CMV, may play a role in pulmonary arterial
disease in immunocompromised children; however, the mechanism for this
potential interaction is unclear.
Herein, we show that pediatric LIP is a multifaceted disease with infiltrative and proliferative components. Infiltration of CD8+ T lymphocytes into the pulmonary interstitium was highly correlative with VCAM-1/VLA-4 adhesive interactions between pulmonary endothelium and blood leukocytes. In addition, individuals with arteriosclerotic lesions demonstrated multifocal aggregates of B cells that occurred in association with high levels of B cell infection with EBV. Although HIV infection and AIDS were requisite for the condition of LIP, the mechanism(s) of induction of VCAM-1 and role of EBV, as a viral co-factor or simply as an opportunistic agent, requires further investigation.
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Acknowledgements |
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Footnotes |
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Supported by funds from the National Institutes of Health, NIAID (AI36613, AI41535, and AI30731).
Accepted for publication February 3, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 integrin interactions. Am J Pathol 1994, 144:27-40[Abstract]
4 integrin by CD4 T cells is required for their entry into brain parenchyma. J Exp Med 1993, 177:57-684ß1-integrin under flow conditions. Blood 1997, 89:3837-38464ß1 integrin. Nature 1992, 356:63-66[Medline]
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-herpesvirus 68 causes severe large-vessel arteritis in mice lacking interferon- responsiveness: a new model for virus-induced vascular disease. Nature Med 1997, 3:1346-1353[Medline]
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