Departments of Medicine and Pathology, George Washington University Medical Center, Washington, DC and Department of Medicine, West Palm Beach Veteran's Administration Medical Center, West Palm Beach, FL, USA
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Abstract |
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Methods. We measured levels of chemokines, IFN-, IFN-
receptor and non-polymorphic MHC Class II protein by high performance capillary electrophoresis, and incubation with antibodies for quantification by chemiluminesce in renal tissue of patients with HIVAN, compared with tissue without HIV infection, in the presence and absence of nephropathy. Renal biopsy tissue protein levels were correlated with the number and type of infiltrating tissue immune cells.
Results. Mean renal interstitial and glomerular MCP-1, RANTES and IL-8 tissue levels were higher in patients with HIV infection compared with tissue without HIV infection, regardless of the presence of renal disease. In contrast, mean renal interstitial and glomerular non-polymorphic MHC Class II, IFN- and IFN-
receptor protein were higher in patients with HIVAN compared with all other groups. Tissue MHC Class II and IFN-
receptor protein levels did not correlate with immune cellular infiltration in patients with HIV infection and renal disease.
Conclusions. The data suggest an upregulated renal immune microenvironment, capable of antigen presentation, exists in HIVAN. MHC Class II proteins and IFNs, and the capacity to present antigen may be crucial in HIVAN pathogenesis.
Keywords: chemokines; interferon ; interferon
; interleukin-8; major histocompatibility II protein; monocyte chemoattractant protein-1; RANTES
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Introduction |
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HIVAN has a highly characteristic pathologic appearance [1]. Tubular reticular inclusions (TRI) may be commonly detected by electron microscopy in renal endothelial cells [1]. TRIs are not pathognomonic for HIVAN, may be found in other renal diseases such as lupus nephritis, and may be induced in vitro by exposure to interferon (IFN)- [1]. HIVAN is uniformly accompanied by an interstitial immune cell infiltrate [1], particularly striking since HIV infection is characterized by peripheral immune cell depletion. These findings suggest the action of chemokines [2] in renal tissue of patients with HIVAN. The role of chemokines in the pathogenesis of nephropathy has been of interest, since they might induce renal dysfunction independent of the pathologic effects of immune cellular infiltration [2]. In addition, the recognition that chemokines such as Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) inhibit productive infection of immune cells and that several chemokine receptors function as coreceptors for HIV infection of immune cells [2,3], and the finding that the prevalence of mutations in certain of these receptors varies between populations at differential risk for HIV infection [3] has stimulated studies of their expression in renal tissue in the presence or absence of renal disease [4]. Although chemokine receptors in the kidney have been localized primarily to infiltrating immune cells [4], their expression can be detected by molecular and immunochemical methods in renal tissue [5]. Although chemokines may induce renal damage [2], chemokines which act as ligands for HIV-coreceptors, such as monocyte chemoattractant protein-1 (MCP-1) and RANTES, could conceivably interfere with HIV infection of renal and immune cells, and blunt the development of HIVAN [6].
Renal cells can present antigen to T cells [7], through the expression of major histocompatibility complex (MHC) Class II proteins, an event regulated by cytokines, especially IFN- [8]. In animal models, however, the presence of MHC Class II proteins and antigen presentation by renal tubular cells alone is insufficient to produce disease [9]. IFN-
has been linked with the pathogenesis of nephropathy in animal models of autoimmune renal disease and IFN-
/IFN-
receptor interactions and IFN-
receptor function modulate a variety of renal pathologic responses in murine models [10]. A requirement for activation of helper T-cells is the presentation of antigen in the presence of class II MHC proteins [11]. Renal cell antigen presentation is facilitated by IFN-
-induced cell surface expression of MHC Class II molecules [9].
We have developed a highly sensitive technique for measuring localized concentrations of cytokines, chemokines, IFN- receptor and MHC Class II proteins in microdissected renal biopsy tissue samples, using microimmunological techniques [12]. This technique permits accurate distinction between adjacent tissue compartments such as the glomerulus and its surrounding interstitium, the major site of inflammatory activity in HIV-associated renal diseases. We examined previously renal tissue IFN-
l levels in HIVAN [12] and found elevated levels compared with controls. However, T-cell IFN-
production may not accurately reflect antigen presenting cell (APC) responsiveness. Cellular expression of IFN-
receptor protein represents a more sensitive indicator of IFN-
responsiveness, particularly in conjunction with assessment of tissue MHC Class II expression.
We compared glomerular and interstitial samples from biopsies of HIV-infected patients with renal disease with glomerular and interstitial samples from biopsies of uninfected patients with similar nephropathies, to determine the relative importance of the presence of the various tissue immune proteins in HIVAN [1], and whether an association exists between the renal immune cellular infiltrate, level of tissue chemokines, and the capacity for antigen presentation in the kidney and disease, by measuring levels of non-polymorphic MHC Class II proteins, IFN- and IFN-
receptor protein. We hypothesized the capacity for responsiveness to antigen presentation typified by high levels of MHC Class II and IFN-
receptor protein expression would differentiate HIVAN from renal disease unassociated with HIV infection, and from renal tissue of HIV-infected patients without renal disease. The primary analyses we conducted were comparisons of renal biopsy tissue levels of immunoreactants in HIVAN and focal segmental glomerulosclerosis (FGS).
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Subjects and methods |
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Pathological studies
Light and electron microscopy were performed as described previously [13]. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections (36 µm thick), which had been deparaffinized and rehydrated. In order to phenotypically identify the infiltrating immune cell types, the sections were stained with a panel of anti-human monoclonal antibodies, directed against UCHL-1 (CD45), a T-cell marker; OPD4, predominantly a T-helper cell marker; L-26 (CD20), a B-cell marker and PG-M1 (CD68), a macrophage marker (Dako Corporation, Carpenteria, CA), as reported previously [13]. Five fields comprising 0.02 mm2 were examined, per section, at x400 power and the number of positively stained cells counted. The total number of infiltrating cells was calculated as the sum of UCHL-1, L-26 and PG-M1 positively stained cells.
Microdissection of renal biopsy material
Cryostat sections (10 µm) were placed on cold (-5°C) mylar coverslips, stained in 0.01% aqueous cotton blue to aid morphological identification of the tissue, and placed into a pre-cooled microincubation chamber (Narishige, Greenvale, NY). Defined morphological areas (glomeruli or tubular interstitium) were microdissected from the surrounding tissue with a Narishige M-155 glass needle micromanipulator, as described previously [14]. Microdissection using this technique can be limited to five or fewer cells. The presence of immune infiltrating cells for exclusion was assessed by visual examination during the dissection of biopsy material. Following microdissection, the areas of interest were flooded with 25 µl of warm (22°C) buffer (100 mM phosphate buffer, pH 7.0, containing 0.2% NP-40), administered with a Narishige M-6 microinjection system. Recovery of the injected fluid was performed with the same instrument, clarified by centrifical ultrafiltration through either a 30 or a 10 kDa filter at 10 000 g for 10 min in a Beckman Airfuge (Beckman Instruments, Palo Alto, CA), and the filtrate analysed by high-performance capillary electrophoresis (HPCE). Prior to HPCE analysis, the total protein concentration of each sample was measured by direct spectrophotometry at 280:260 nm and normalized to 10 µg protein/ml [15].
High-performance capillary electrophoresis measurement of chemokines in microdissected samples
Approximately 20 nl samples were introduced into either uncoated or polyethylene glycol-coated capillaries, filled with 100 mM phosphate buffer, by vacuum injection and electrophoretically separated at 27 kV constant voltage. The migration of the sample components was monitored by on-line UV detection at 200 nm and the electropherogram directly read into a computerized recording system. Continuous fractions were collected on a linear modification of a circular membrane-based system, using an ImmobilonTM-P polyvinylidene difluoride membrane (Millipore, Bedford, MA), as the collection device. Similar separations were performed using purified or recombinant cytokines as standards.
Measurement of IFN- receptors and MHC Class II protein by high-performance immunoaffinity chromatography (HPIAC)
HPIAC was used to isolate and measure IFN- receptor and non-polymorphic MHC class II proteins from the microdissected tissue samples. Briefly, 10 µm diameter glass beads were coated with streptavidin prior to applying the antibody coat [15]. Monoclonal antibodies raised against IFN-
receptors (R & D Systems, Minneapolis, MN), and non-polymorphic MHC class II molecules (One Lambda, Los Angeles, CA), were modified by attaching biotinamido-caproyl-hydrazide to the carbohydrate moieties in the antibody Fc portion and immobilizing them onto the streptavidin-coated beads via the biotinavidin interaction. The antibody-coated beads were slurry-packed into 50x4.6 mm PEEK HPLC columns (Alltech Associates, Deerfield, IL), producing HPIAC columns containing 125 µg of immobilized antibody. The columns were attached to a Beckman 340 HPLC System, equipped with a Model 112 pump, a Model 160 UV detector (set at 280 nm, 0.5 AUF), and a Shimadzu CR-3B peak integrator. Separations were run in an isocratic mode for 15 min, using a mixture of 0.015 M NaCl/0.1 M C2H3NaO2, pH 6.5 as the running buffer. At 15 min, a 04 M NaSCN linear gradient, controlled with an Autochrom III OPG/S solvent selectorcontroller (Autochrom, Milford MA), was added to the running buffer over a 15 min period and the chromatogram monitored at 280 nm by the peak integrator. Eluted fractions were collected for further analysis.
Chemiluminescent ELISA-immunoassay measurement of HPCE and HPIAC isolated fractions
Specific protein or peptide concentrations in fractions collected from each HPCE or HPIAC separation were measured by chemiluminescence-enhanced enzyme linked immunoabsorbent assay (CHEM-ELISA) [15], using specific alkaline phosphatase-labelled antibodies, directed against interleukin (IL)-8, MCP-1, RANTES and IFN- (Genzyme Corporation, Cambridge, MA). Briefly, the membrane was removed from the fraction collector and divided into strips. Each strip was incubated with a specific enzyme-labelled antibody, directed against the chemokines of interest, for 24 h at room temperature. The strips were washed in 100 mM phosphate buffer, pH 7.2 and the bound antibodies detected following their reaction with 3-[2'-spiroadamantane]-4-methoxy-4-[3''-phosphoryloxy]phenol-1,2-dioxetane (AMPPD), an alkaline phosphatase chemiluminescence substrate (Tropix, Bedford, MA). The CHEM-ELISA results were analysed by the ANELISA-R software package (Man-Tech Associates, Buffalo, NY).
Statistical analysis
Primary comparisons were made between tissue levels in biopsies of patients with HIVAN and idiopathic FGS by unpaired t-tests. In other analyses, differences among groups were assessed by ANOVA, paired or unpaired Student's t-test, or Fisher's PLSD test as appropriate. Correlations between patients' CD4 counts and CD4/CD8 ratios at the time of biopsy, the total number of interstitial and glomerular immune cells, and total number of interstitial and glomerular T-helper cells and macrophages, and glomerular and interstitial levels of IL-8, RANTES, MCP-1, non-polymorphic MHC Class II protein, IFN- and IFN-
receptor levels were performed in the group with HIVAN. Secondary analyses were conducted of these correlations in tissue from patients with FGS. P<0.05 was taken as the level of significance. Data are reported as mean±SEM.
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Results |
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Levels of IL-8, RANTES and MCP-1 were greater in interstitial compared with glomerular tissue in uninfected patients with FGS, but there was no difference between mean interstitial and glomerular tissue chemokine levels measured in patients with HIVAN. Correlations were performed assessing the relationship of time between acquisition of autopsy material and glomerular and interstitial levels of IL-8, RANTES and MCP-1. There was no correlation of time and tissue levels of glomerular or interstitial MCP-1 or RANTES. Only the correlation of time and tissue levels of glomerular (but not interstitial) levels of IL-8 achieved the level of statistical significance. There was no difference between storage times for autopsy material from HIV infected and uninfected subjects without renal disease (Groups III and IV).
Patterns of expression of IL-8, RANTES and MCP-1 were similar in the pathologic groups (Tables 1 and 2
). Mean interstitial and glomerular chemokine levels were higher in HIVAN tissue compared with both idiopathic FGS (unpaired t-test) and uninfected renal tissue in the absence of nephropathy (Groups I and IV, ANOVA) (Tables 1
and 2
). In secondary analyses analysing the control autopsy tissue, mean renal tissue chemokine levels were higher in Group III (patients with HIV infection who died without clinical or pathologic evidence of renal disease) compared with tissue from patients in all other groups (Tables 1
and 2
). Mean renal tissue chemokine levels were lower in uninfected (Groups I and IV) when compared with HIV-infected groups (Groups II and III) (ANOVA).
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Correlations were performed assessing the relationship of time between acquisition of autopsy material and glomerular and interstitial levels of MHC Class II protein, IFN- receptor protein and IFN-
. There was no correlation of time and tissue levels of glomerular or interstitial MHC Class II protein, IFN-
receptor protein and IFN-
.
CD4 counts in Group II patients at the time of biopsy correlated with glomerular IL-8 (r=0.903, P<0.015) levels. The association between CD4 count and glomerular MCP-1 levels in the six Group II patients approached the level of significance (r=0.787, P=0.063). Interstitial IL-8 levels correlated with patients' CD4/CD8 ratio (r=0.841, P<0.04), while correlations with total interstitial immune cell number and glomerular MCP-1 levels with the CD4/CD8 ratio approached the level of significance (r=0.783, P=0.066 and r=0.762, P=0.078, respectively).
In biopsy material from patients with HIVAN, glomerular IL-8 and IFN- levels correlated with the total number of glomerular immune cells, glomerular macrophages and interstitial T-helper cells (Table 4
). Glomerular total T-helper cell number correlated with the level of interstitial MHC Class II protein (r=0.903, P=0.036). There was no significant correlation between renal interstitial or glomerular chemokine, MHC Class II or IFN-
or IFN-
receptor protein levels and the total number of interstitial immune cells or macrophages in this group of patients, although in some analyses the r values were in the range of 0.50.7. Interestingly, in contrast to the few relationships between immunoreactants and cell populations in HIVAN biopsy tissue, in biopsies from the slightly larger group of patients with FGS, there were significant correlations between interstitial tissue levels of RANTES, MCP-1, IFN-
, IFN-
receptor and MHC Class II protein levels and interstitial total and macrophage cell number, and between glomerular tissue levels of IL-8, RANTES and MCP-1 and glomerular total and macrophage cell number (data not shown).
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Discussion |
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However, T-cell infiltration and subsequent activation in the setting of HIV infection may not result in recruitment of effector mechanisms. In HIV infection, T-cell activation may result in cell death rather than proliferation [11]. We showed increased apoptosis in renal tubular tissue of patients with HIVAN compared with similar tissue from patients with idiopathic FGS in the absence of HIV infection [16]. HIV-1 proteins may have cytopathic effects on renal cells, which could enhance nephrocytotoxicity (reviewed in Shukla et al. [17]).
We found increased levels of IL-8, RANTES and MCP-1 in HIVAN, compared with interstitial and glomerular tissue from patients with idiopathic FGS. Although autopsy renal tissue from uninfected patients without nephropathy showed low tissue chemokine levels, surprisingly, tissue from HIV-infected subjects without clinical evidence of renal disease was characterized by levels of IL-8, MCP-1 and RANTES greater than those in HIVAN. One interpretation of the data suggests HIV-infected patients' renal tissue exists in a microenvironment characterized by high chemokine levels, perhaps as a host defense response. Alternatively, a robust renal cellular chemokine synthetic response may limit productive renal and immune cellular HIV infection and therefore limit the possibility of developing HIVAN [1,3,6]. Renal tissue chemokines (especially if unable to bind with their receptors), like the presence of HIV genome in the kidneys of HIV-infected subjects [14], are insufficient alone to explain the development of nephropathy.
We investigated, in other studies, renal tissue levels of chemokine receptors CCR-3 and CCR-5, along with MCP-1 and RANTES in biopsy tissue of 11 patients with HIVAN and 19 with idiopathic FGS. There were no differences between interstitial or glomerular chemokine or chemokine receptor levels in patients with FGS and HIVAN. We did not assess CXCR4 or proteins involved in mediating apoptosis in these studies. These preliminary findings, however, emphasize that the relationship between chemokine levels and their receptors and the pathogenesis of disease is not yet well established.
We also acknowledge that autopsy tissue, in part because of its different preparation, is not exactly comparable with biopsy tissue. We have successfully used autopsy material for comparative purposes in previous studies. However, in the analyses of the chemokines in this study, autopsy tissue in patients without clinical renal disease showed widely divergent levels in the presence and absence of HIV infection, demonstrating that the results are not simply artifactual, or a consequence of differences in sample presentation.
This is to our knowledge the first proteomic study of renal tissue immunoreactants in patients with renal disease in the presence and absence of HIV infection. We did not use special buffers to inhibit RNA degradation, nor did we assess mRNA associated with the studied proteins, which must be considered in assessing the findings. It is unlikely, however, that nascent mRNA is engaged in active protein synthesis during tissue processing and storage. Although autopsy tissue was acquired at different times after death and analysed at variable periods after autopsy, there was, however, no correlation of storage time and tissue levels of IFN-, IFN-
receptor and MHC Class II proteins. Future studies of renal tissue will benefit from the assessment of both genomic and proteomic factors in well characterized and preserved tissue processed under standardized conditions, as well as careful determination of the relationship between acquisition and storage time and analyte levels.
T-cell activation depends on the effective interactions of the T-cell receptor with MHC proteins and antigen on the APC [8,11], but costimulatory cellpeptidecell interactions must also be present to activate T-helper cells [8]. The presence of either a HIV peptide or neoantigen could act as a costimulatory signal, eliciting active T-cell responses, rather than producing anergy. The induction of renal glomerular and tubular epithelial cells into effective APCs [7] may be pivotal in the pathogenesis of HIVAN. The ability of renal cells to present antigen, especially in response to IFN-, and express intracellular adhesion molecules and proinflammatory cytokines allows them to participate in a tissue immune response [11]. Increased expression of MHC Class II protein is present in murine lupus nephritis and human renal allograft rejection as well as other renal diseases [18]. However, increased expression of MHC Class II protein in a transgenic model was insufficient alone to result in nephropathy [19], highlighting the necessity of additional factors, such as the presence of a nephritogenic antigen, for disease pathogenesis.
Tissue MHC class II and IFN- receptor protein levels were higher in HIVAN than the other three groups. This was not the case for the tissue chemokines, suggesting MHC Class II protein and IFN-
receptor expression are specifically and highly upregulated in renal tissue of HIV-infected patients with HIVAN. However, these increased tissue protein levels were not correlated with the increased immune cell infiltration seen in HIVAN. The lack of correlation between these parameters also suggests that MHC Class II and IFN-
receptor protein expression are not related solely to tissue immune cell infiltration, but rather to renal cellular responses. IFNs may upregulate MHC protein in the kidney, and can modulate mesangial cell function [9,10]. Binding of IFN-
to its receptor may facilitate presentation of antigen not only by renal tubular and glomerular cells, but by inducing MHC Class I and II antigen expression in macrophages and endothelial cells, and activating monocytic cells [8,10,11]. IFN-
, the product of virally infected cells [8], has pleiotropic effects on cells within its microenvironment. The association of IFN-
specifically with HIVAN in this study is intriguing in light of its pathogenic association with TRIs, a common feature of HIVAN [1]. In addition, the administration of IFN-
and IFN-
has been associated with the development of renal disease, including FGS, in animal models and in humans [8,20].
Our data specifically implicate MHC class II protein expression, IFN- and the interaction between IFN-
and its receptor in the pathogenesis of HIVAN. Upregulation of MHC Class II and IFN-
receptor protein expression in renal tissue of HIVAN is consistent with an immunologically activated renal microenvironment involving T-cells, HIV peptides and renal parenchymal cells, as well as cytokines and chemokines. The ability of renal parenchymal cells in the interstitium and glomeruli to express MHC Class II protein may facilitate renal cell cytotoxicity. Alternatively, in the absence of the proper costimulatory signals, interaction of T cells and APCs, or the effects of IFNs might result in apoptosis of immune and epithelial cells [11], a characteristic feature of HIVAN [1,16]. Therefore, increased MHC Class II expression and the action of IFNs in the renal microenvironment may facilitate the pathogenesis of HIVAN by two mechanisms.
The particular cytokine profile in an activated, receptive renal microenvironment, however, might be critical in mediating immune and fibrogenic outcomes. Chemokines that attract macrophages and amplify T-cell responses may result in a pathologic effector response, but only in susceptible patients. Such chemokines may play a role in mediating renal tissue damage by infiltrating lymphocytes and mononuclear cells, after an initial initiating event, such as the infection of a renal cell by HIV or the infiltration of an HIV-infected lymphocyte or monocyte in renal tissue. Their action probably depends on unimpeded interactions with their receptors in tissue. Virus-induced differences in MHC protein or other host protein expression may also be critical in the pathogenesis of the disease.
This was a relatively small clinicalpathologic correlative study, using tissue acquired by biopsy, surgery and autopsy. However, the analyses using surgical and autopsy tissue provide intriguing preliminary data regarding differences that should be pursued in control tissue acquired in an identical manner in future studies. The hypotheses generated in this study regarding the role of the immunoreactants in the pathogenesis of HIVAN must be further and separately evaluated in in vitro systems to assess the individual and synergistic effects of these processes on renal cellular function.
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Acknowledgments |
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Notes |
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References |
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