Chlamydia pneumoniae has been isolated as a third species of the intracellular pathogen Chlamydia genus and recognized as a common respiratory pathogen (1–5). Infection with C. pneumoniae seems to be almost obligate at least once in a lifetime, and reinfections with this organism are common (5). More recently, C. pneumoniae has been implicated in the pathogenesis of atherosclerosis and acute coronary syndromes, first in Finland (6) and then later in several other countries (7–12). Endovascular detection of the microorganism has been demonstrated by a wide variety of methods in the atherosclerotic plaques (5).
In the general population, infection is absent in children under 5 years of age, and a peak incidence of 9.2% occurs between the ages of 5 to 10, falling back to 1.5% above the age of 20 (12). It has been suggested that adult infections are mainly reinfections characterized by specific immunoglobulin (Ig) G and IgA antibody responses without changes in IgM (12, 13).
The foam cell induction ability of C. pneumoniae has been reported, and several groups have described this microorganism in foam cells of human atherosclerotic lesions (14), experimental animals (12, 15), and in vitro (7, 16). Foam cell arteriopathy or chronic obliterative arteriopathy is one of the generally accepted pathologic features of chronic liver allograft rejection (17–19), which has several similarities to the early lesions in atherosclerosis. Infection-reinfection or exacerbation is a serious undesirable side effect of immunosuppressive therapy in patients undergoing organ transplantation (20). This raises the issue that possible C. pneumoniae infection or exacerbation might play a role in foam cell arteriopathy during chronic rejection.
MATERIALS AND METHODS
Patients
Ten patients showed chronic liver allograft rejection (CLAR 1–10), which slowly progressed despite high-dose azathioprine therapy; therefore, retransplantation was performed at the end stage of the rejection liver disease. Tissue samples were taken for histology, immunohistochemistry, and solution-phase polymerase chain reaction (PCR) from the explanted livers during the process of retransplantation. Liver samples showing histologic evidence of foam cell arteriopathy were analyzed by immunohistochemistry and PCR. Age- and sex-matched control liver samples were obtained shortly after death from 10 individuals who died unexpectedly because of some sort of an accident (control liver samples=CL 1–10) (Table 1).
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Table 1: Summary of the patients' data, with results of Chlamydia pneumoniae PCR and immunohistochemistry assays
Liver samples from two transplant patients with chronic liver rejection without foam cell arteriopathy and from nine liver transplant patients with acute allograft rejection served as special controls. This study was carried out according to the guidelines of the Helsinki Declaration on human rights.
Histology
Frozen and paraffin-embedded sections were prepared and stained by hematoxylineosin.
PCR
C. pneumoniae PCR. Total nucleic acid was isolated by Masterpure Complete DNA and RNA Purification Kit (Epicentre Technologies, Madison, WI). One gram of frozen tissue samples was homogenized by sterile tissue grinder (Kontes, Vineland, NJ) in 5 mL of phosphate-buffered saline (PBS) (pH 7.4). To the 25 μ L (~5 mg) of the homogenized tissue, 300 μ L of Tissue and Cell Lysis Solution containing 100 μ g proteinase K (Sigma Chemical, St. Louis, MO) was added. This solution was mixed thoroughly by vortex, incubated, and then continuously shaken at 65°C for 40 min in an Eppendorf Thermomixer 5437. The microcentrifuge tubes (Greiner Labortechnik Gmbh, Frickenhausen, Germany) were then placed on ice for 5 min and 150 μ L of MPC Protein Precipitation Reagent was added into each one. After a centrifugation step (10,000 g; 10 min; 4°C), the supernatant was transferred to a new microcentrifuge tube and 500 μ L of isopropanol (Merck KGaA, Darmstadt, Germany) was added. After centrifugation (10,000 g; 10 min; 4°C), the pellet was rinsed twice by addition of 500 μ L of 75% ethanol (Merck KGaA, Darmstadt, Germany), followed by a centrifugation step (10,000 g; 10 min; 4°C). Then, the pellet was air-dried and the nucleic acids resuspended in 35 μ L of Tris-EDTA buffer (pH 8.0). From each sample, the concentration of DNA was determined by measuring the optical density on 260-nm wavelength (Pharmacia Biotech GeneQuant II, Uppsala, Sweden). Single-step solution-phase PCR with C. pneumoniae-specific primers CpnA sense (TGA CAA CTG TAG AAA TAC AGC) and CpnB antisense (CGC CTC TCT CCT ATA AAT) was carried out as described by Gaydos et al. (21).
One microgram of extracted DNA was amplified by a master mix containing 0.5 μ M primers; 0.2 μ M deoxynucleoside triphosphate mix (Promega Corporation, Madison, WI); 1×PCR buffer II. (Perkin Elmer Applied Biosystems, Foster City, CA); 2.5 mM MgCl2 (Perkin Elmer Applied Biosystems); 0.01% gelatine (Sigma Chemical) in a total volume of 50 μ L in Costar 0.5 mL PCR tubes (Corning Inc., Science Products Division, Acton, MA). Ten-minute denaturation (94°C); 35 cycles of denaturation (94°C, 1 min), annealing (56°C, 1 min), then elongation (72°C, 1 min); and 10 min exhaustion (72°C) were performed on the samples in a Hybaid OmniGene thermocycler (Hybaid Limited, Ashford, Middlesex, United Kingdom). One hundred microliters of mineral oil (Sigma Chemical) was used to prevent evaporation of reaction mix during the cycles. Ten microliters of PCR products from each sample and 0.85 μ L of 100-base pair molecular size standard (Life Technologies, Inc., Rockville, MD) were mixed with 3 μ L of gel loading solution (G2526; Sigma Chemical) and loaded in a 2% agarose gel.
Nucleic acids were extracted and C. pneumoniae-specific sequences were amplified from all tissue samples in two independent series. Positive and negative controls were included in each assay. The negative controls contained all PCR reagents and sterile distilled water. Human vascular endothelial cell line cells infected in vitro with C. pneumoniae were used as a positive control. Serial dilution of purified C. pneumoniae DNA was included for all runs to ensure successful nucleic acid amplification. To avoid the risk of contamination, sample preparation, PCR amplification, and product analysis were performed in separate rooms.
Cytomegalovirus PCR.
The DNA extraction procedure is the same as described above for the C. pneumoniae PCR. The cytomegalovirus (CMV) PCR was carried out in a Hybaid PCR-Express thermal cycler. Primers targeting the glycoprotein B gene of human cytomegalovirus (final product 149 base pairs) were used in the reactions with the following sequences:
Forward primer, 5′-GAGGACAACGAAATCCTGTTGGGCA-3′.
Reverse primer, 5′-GTCGACGGTGGAGATACTGCTGAGG-3′.
One microgram of extracted DNA was added to the PCR cocktail reagents in a reaction tube prewarmed to 70°C and the PCR cycling started. The positive controls were tissues previously testing positive for CMV. The cycling parameters were set for 40-step cycles with 30 s denaturation at 94°C, 30 s annealing at 60°C, and 45 s extension at 72°C followed by a final extension at 72°C for 10 min. The amplified products were analyzed using 2% agarose gel electrophoresis at 100 V for 30 min, stained with ethidium bromide, and visualized under UV light.
Chlamydia pneumoniae-Specific Monoclonal Antibody Staining
Ten samples from patients with chronic liver rejection (CLAR 1–10) were examined initially for evidence of chlamydial infection, nine samples from individuals with negative PCR results for chlamydia (CL 1, 3–10) were used as negative, and 1 PCR-positive sample (CL 2) was used as positive control. Hep-G2 cells from cell culture infected with C. pneumoniae were also used as a positive control. Different assays for immunofluorescent-immunohistochemical detection of chlamydial antigens were applied as follows.
First, frozen sections were acetone-fixed (−20°C) for 15 min for an immunofluorescent staining procedure. After fixation, the sections were air-dried for 30 min and then stored frozen at −70°C until use. For the direct immunofluorescent technique, RR 402 C. pneumoniae-specific monoclonal antibody conjugated with fluorescein isothiocyanate (FITC) (IMAGEN Chlamydia pneumoniae FITC Research Reagent K6601; Dako Corp., Carpinteria, CA) was used on the samples in 1:200 dilution. Then, in the indirect immunofluorescent method, RR 402 C. pneumoniae-specific monoclonal primary antibody (Washington Research Foundation, Seattle, WA) was used on the sections overnight at 4°C in 1:400 dilution. Then, the specifically bound primary antibodies were visualized by fluorescein isothiocyanate-conjugated secondary antibody (F 5387; Sigma) (Fig. 3a).
Figure 3: Indirect immunofluorescent detection of Chlamydia pneumoniae in a chronic rejection liver sample with arteriopathy (CLAR sample) using monoclonal RR 402 antibody. Two elementary bodies (white spherical bodies) are localized in a perisinusoidal cell (arrow) (magnification, ×1,250). (a) Immunohistochemical detection of Chlamydia pneumoniae in the CL 2 (b) and in another (c) control sample. CL 2 is the only PCR-positive sample from the controls obtained shortly after death from the 10 individuals who died unexpectedly because of accidents. Positive signals (black dots which mostly form small clusters) are mainly seen in periportal areas, in the hepatocytes. Moreover, positivities are occasionally localized in the sinusoidal lining cells (arrows) (b). The other CL samples showed no specific positivity (c) (RR 402 monoclonal antibody with substrate system, DAKO LSAB+–AP and new fuchsin as chromogen [b] ×500 and [c] ×250).
The samples were analyzed by fluorescent microscopy and a confocal laser scanning microscope (BioRad MRC-1024). The presence of three or more elementary bodies per sample with characteristic morphology was regarded as a positive result.
Second, immunohistochemistry on paraffin-embedded samples was performed. To retrieve antigens after the standard procedures of formalin fixation, embedding, cutting, and deparaffinization, tissue sections were treated by microwave in Antigen Unmasking Solution (Vector Laboratories, Inc., Burlingame, CA) for 5 min. RR 402 C. pneumoniae-specific monoclonal primary antibody (Washington Research Foundation, Seattle, WA) was used overnight at 4°C in 1:400 dilution on the samples with the following visualization systems: (1) DAKO LSAB+–alkaline phosphatase (K 0689, Dako Corp., Carpinteria, CA, USA)–New Fuchsin (K 0698, Dako Corp., Carpinteria, CA, USA) (Fig. 3b and c); (2) DAKO ENVISION–Alkaline phosphatase–Fast Red (K 4016, Dako); and (3) DAKO LSAB2–horseradish peroxidase (K 0675, Dako)–AEC (HK 129-5K; BioGenex, San Ramon, CA) (Figs. 2a and b, 4, and 5).
Figure 2: Immunohistochemical detection of Chlamydia pneumoniae in samples from patients with chronic liver allograft rejection showing histologic evidence of foam cell arteriopathy (CLAR samples). Strong Chlamydia pneumoniae positivity (black dots) was detected in the hepatocytes (a). The distribution of positive hepatocytes shows centrolobular predominance (b) (RR 402 monoclonal antibody and DAKO LSAB2–horseradish peroxidase detection system with AEC as chromogen; magnification ×500).
Figure 4: Immunohistochemical detection of Chlamydia pneumoniae in samples from patients with chronic liver allograft rejection showing foam cell arteriopathy (CLAR samples). Occasionally, positive (black dots) signals were detected in portal tracts (a). Other portal regions were infiltrated with histiocytes showing strong Chlamydia pneumoniae positivity (b). Perivascular positivity around the portal small blood vessels was also observed (c) (RR 402 monoclonal antibody and DAKO LSAB2–horseradish peroxidase detection system with AEC as chromogen; magnification, ×500).
Figure 5: Immunohistochemical detection of Chlamydia pneumoniae in chronic obliterative arteriopathies. Usually, the altered hepatic arteries were negative or showed occasionally positivity (black dots), localized mainly in the adventitial cells of the arterial wall, whereas the foam cells were negative. (a) Very early lesion. Two foam cells in the intima, one of them covered with a positive endothelial cell (arrow); one of the adventitial cells shows positivity (double arrow). (b) Complete lesion: obliterative foam cell arteriopathy with a positive adventitial cell (arrow). (c) Special lesion: the arterial lumen is obliterated by granulation tissue-like mass containing no foam cells but capillaries, fibroblasts, and histiocytes. Some of these cells show strong positivity (RR 402 monoclonal antibody and DAKO LSAB2–horseradish peroxidase detection system with AEC as chromogen [a] ×1,250×; [b] ×500; [c] ×250) (a and b, differential interference contrast–Nomarsky).
The samples were analyzed by brightfield microscopy and scored semiquantitatively. Only strong signals with characteristic color and morphology were regarded as positive.
RESULTS
Histology
All samples taken from the 10 transplanted livers with foam cell arteriopathy (CLAR 1–10) showed the following pathologic alterations: chronic arteriopathy, vanishing bile duct syndrome, cholestasis, and fibrosis. Other relevant pathogens were excluded by light microscopy examination.
Detection of C. pneumoniae DNA by PCR
Single-step solution-phase PCR was performed to detect chlamydial DNA. In all 10 chronic rejection liver samples (CLAR 1–10), C. pneumoniae DNA was detected (Fig. 1a and Table 1). Because all 10 samples were repeatedly found positive (while the extraction controls were negative), it may be stated with confidence that none of the reactions were false-positive because of PCR amplicon contamination.
Figure 1: (a)
Chlamydia pneumoniae PCR results of chronic liver allograft rejection samples. Lanes 1 and 16, 100-base pair DNA ladder. Lanes 2 and 15, empty. Lanes 3 and 12, CLAR 1 to 10. Lane 13, positive control. Lane 14, negative control. All the lanes of CLAR samples and the lane of the positive control contain the 465-base pair specific band. The lane of the negative control contains no band. (b)
Chlamydia pneumoniae PCR results of control samples. Lanes 1, 18, 19, 29, 100-base pair DNA ladder. Lanes 2 and 28, negative controls. Lanes 3 to 12, control liver samples (CL) 1 to 10. Lanes 13 and 14, chronic rejection liver samples without any evidence of arteriopathy. Lanes 15 and 16, acute rejection liver samples. Lanes 17 and 27, positive controls. Lanes 20 to 26, acute rejection samples. The lanes of positive controls and CL 2, moreover, one lane (
25) of the acute rejection liver samples contain the 465-base pair specific band. All the other lanes of CL and the lane of negative control contain no band.
Of the 10 control samples from individuals who died because of some sort of accident (CL 1–10), 1 sample was found positive for chlamydial DNA, as was also 1 from the 9 liver samples from patients with acute liver allograft rejection. Both chronic liver rejection samples without foam cell arteriopathy were found to be negative (Fig. 1b).
Detection of Cytomegalovirus DNA by PCR
Five of 10 CLAR samples showed CMV positivity, namely, CLAR 1, CLAR 2, CLAR 4, CLAR 6, and CLAR 9. All of the 10 control samples (CL 1–10) were negative for CMV.
Detection of C. pneumoniae Antigens by Specific Monoclonal Antibody Staining
The 10 control liver samples from individuals who died because of accidents (CL 1–10) as well as the 10 chronic rejection liver samples (CLAR 1–10) were stained with C. pneumoniae-specific antibodies (Table 1). The nine PCR-negative control samples (CL 1, 3–10) failed to show specific staining, whereas all the chronic rejection samples (CLAR 1–10) and one PCR-positive control sample (CL 2) contained specific, apple-green-fluorescing chlamydial elementary bodies by the fluorescent technique (Fig. 3) and showed specific positivity by immunohistochemical stainings.
Positive signals were localized mainly in the hepatocytes (Fig. 2a), mostly with involvement of the centrolobular region (Fig. 2b). Several sinusoidal and perisinusoidal cells as well as some of the endothelial cells also showed positivity (Fig. 3a and b). Usually, the portal regions were negative. In some cases, however, positive signals were detected in cells of portal tracts (Fig. 4a), especially in one sample where numerous positive cells corresponded probably to portal tract histiocytes (Fig. 4b). In another case, perivascular positivity around small blood vessels was also observed in the portal region (Fig. 4c).
Moreover, the altered hepatic arteries were negative or showed occasional positivity seen localized mainly in the adventitial cells of the arterial wall, whereas the foam cells were negative (Fig. 5a and b). However, a special form of obliterative arteriopathy was also present in one of the samples. In this type, the arterial lumen was obliterated by a granulation tissue-like mass containing no foam cells but capillaries, fibroblasts, and histiocytes. Several cells of the obliterating tissue showed a strong positivity (Fig. 5c). In the PCR-positive control liver (CL 2), positive signals were detected usually in periportal localization (Fig. 3b), in contrast to the chronic liver rejection samples where the positive signals were found mainly localized in the centrolobular region.
DISCUSSION
The cause of chronic arteriopathy—a severe complication of chronic rejection after orthotopic liver transplantation—has not yet been properly clarified. Since the early 1970s, more and more reports have called attention to the connection between rejection and certain infectious agents. The available data primarily referred to the role of herpes viruses (CMV, herpes simplex, and zoster) and Listeria monocytogenes. Later, on the basis of epidemiologic surveys, the acute rejection-causing effect of the influenza B virus and the adenoviruses had been raised. A connection was found between chronic liver rejection and CMV infection, also supported by results from animal experiments as well as human studies. There are several mechanisms by which CMV can promote the development of chronic transplant dysfunction-related vascular damage. The infection increases the intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and lymphocyte function associated antigen-3 molecules on the endothelial and other infected cell surfaces, which in turn enhances migration of inflammatory cells as well as activates T cells and their interferon-γ production, contributing to the direct effect of the virus. The smooth muscle cells start to proliferate, the endothelial cells become damaged by the effect of the local inflammation, and the ineffective repair can lead to plaque development. Furthermore, there is sequence homology between the CMV and the human leukocyte antigen (HLA)-I heavy chain; thus, the viral infection can enhance the antiallograft T-lymphocyte cytotoxicity. Sequence homology is also present between the viral immediate-early region and the HLA-DR β-chain which, by means of cross-reactive antibodies, can lead to the damage of HLA-DR–expressing allograft blood vessels (22).
The pathogenetic role of C. pneumoniae in the lesions of arterial walls is also widely supposed. Detection of endovascular chlamydial antigen by immunohistochemistry has been found to vary between 42% and 100% in atherosclerosis (23), whereas others have only found the chlamydial presence in the vessels to be approximately 15% to 26% (5). This lower rate of positive cases was explained by some technical differences; in fact, immunostaining usually produces the highest positivity rate (5, 12, 24). The reported differences in rates might also be associated with different geographical regions and the localization of the arteries, namely, central or peripheral (5). Chronic rejection arteriopathy is another alteration where the occurrence of C. pneumoniae has been proved and may be considered as a potential factor in the pathogenesis (25). Questions similar to those arising with respect to arteriosclerosis are whether C. pneumoniae is actually an initiating factor or facilitates the progression, or it is only a coexisting nonpathogen in chronic rejection arteriopathy.
Our study proved first the presence of C. pneumoniae DNA by PCR in the transplanted, chronically rejected livers. We were able to confirm the presence of C. pneumoniae major outer membrane protein antigen by immunohistochemistry in different cellular localizations. All 10 cases of chronic liver allograft rejection with foam cell arteriopathy (CLAR 1–10) were positive for C. pneumoniae infection, contrary to the 1 out of 10 age- and sex-matched controls (CL 1–10). By comparison, the results of both PCR and immunohistochemistry were in agreement. The good correlation between the PCR method and the antigen detection methods supports the proposal that these liver tissues are truly infected and the PCR results do not simply represent remnant pieces of nucleic acid. It would be of importance to confirm the viability of these infections by cell culture or reverse-transcriptase PCR approaches, because the DNA (not reverse) PCR is considered as an established method for identifying intact chlamydia (5); it cannot, however, differentiate between replicating and nonreplicating organisms.
We localized C. pneumoniae-specific antigen by immunohistochemistry both in altered tissue components and in such hepatic arteries, sinusoidal endothelial cells, perisinusoidal cells, and hepatocytes that seemed to be intact. This is in agreement with studies showing that C. pneumoniae-positive tissues did not necessarily show prominent inflammation (5). C. pneumoniae was found to be localized in hepatic vessels whether or not showing significant alterations, suggesting that C. pneumoniae is merely a secondary pathogen passenger (23, 26). In one of the samples, we also found that some of the arterial lumens were obliterated by a granulation tissue-like mass without any foam cells, but containing capillaries, fibroblasts, and histiocytes. Several cells of the obliterating tissue showed strong positivity. This type of alteration might be a special different kind of arterial lesion or might represent another stage of obliterative arteriopathy.
The high ratio of C. pneumoniae positivity in chronic rejection might be the result of exacerbation of a previous infection with the bacteria because of immunosuppressive therapy. However, our preliminary results suggest that besides the immunosuppression, there should also be other factors that affect the detectable presence of C. pneumoniae in transplanted livers. These data have shown that C. pneumoniae is mostly undetectable in acute liver allograft rejection, as well as in chronic liver allograft rejection in which foam cell arteriopathy is lacking (Fig. 1b). Therefore, the presence of arteriopathy might be one of the important factors offering a reason for the detected high frequency of C. pneumoniae in chronic liver allograft rejection samples. In contrast to the 100%C. pneumoniae positivity, the presence of cytomegalovirus by PCR was detectable in only 50% of CLAR samples, referring to the fact that the connection—if any—between the development of CMV infection and chronic arteriopathy is much weaker than in case of C. pneumoniae.
It has been shown that endothelial cells, smooth muscle cells, and macrophages can be infected in vitro with C. pneumoniae (12, 27). Human macrophages infected with C. pneumoniae and incubated with low-density lipoprotein have been shown to transform into foam cells (28). The fact that C. pneumoniae is able to produce changes in endothelial cells, smooth muscle cells, and macrophages in vitro suggests that the cellular alterations as the result of C. pneumoniae infection can occur in vivo. This is supported by our results strongly suggesting that C. pneumoniae is associated with foam cell arteriopathy after transplantation. The immunohistochemical investigations have shown just the arterial foam cells to be negative for C. pneumoniae antigens. A possible explanation for this could be provided by the theory that the cross-reaction between chlamydial hsp60 antibodies and human hsp60 stands partially in the background of C. pneumoniae-related arterial changes. Presumedly, the auto-immune inflammation triggered in such manner after sensibilization can progress in the minimal presence or even the absence of the chlamydial antigen, in which oxidized low-density lipoprotein—also behaving as an autoantigen—can play a role (29). Mention should be made, however, of a recent publication, the results of which seem to contradict the fact of cross-reaction between chlamydial and human hsp60 (30). Nevertheless, there is a study that can probably offer another possible answer to resolve this problem. In fact, Blessing et al. found that the growth of C. pneumoniae was significantly inhibited by lipid loading of macrophages (31). This may indicate that the chlamydial infection can induce formation of foam cells, but the bacteria in fully developed foam cells might decrease to a much lower level.
CONCLUSION
Therefore, although the immunohistochemical negativity of foam cells is apparently inconsistent with a supposed pathogen role of C. pneumoniae in this form of arteriopathy, our observations strongly suggest that the considerable presence of this bacterium is associated with the occurrence of arteriopathy in chronic liver rejection. However, because these results reflect only the end stage of a chronic rejection process showing slow progression in highly immunosuppressed patients, further studies are necessitated to specify exactly whether C. pneumoniae is a primary or secondary pathogen (as suggested in atherosclerosis), or is only a coexisting nonpathogen in chronic rejection. Accordingly, investigations are still needed to clarify the exact role of chlamydial infection in the pathogenesis of chronic rejection arteriopathy, including studies on the effectiveness of a potential antibiotic treatment in the inhibition of lesion development.
Acknowledgments.
The authors thank Ágnes Szik and Krisztina Egedy for technical assistance.
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