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Departments of Neurology and Pathology, Vanderbilt University Medical Center, Nashville, Tennessee
To examine a possible relationship between Chlamydia pneumoniae infection and multiple sclerosis (MS), we undertook an immunohistochemical (IHC), molecular, and ultrastructural comparison of central nervous system (CNS) tissue and cerebrospinal fluid (CSF) sediment from patients with MS and control individuals with other neurological diseases (ONDs). In 7 of 20 MS cases, IHC staining was seen in association with ependymal surfaces and periventricular regions of formalin-fixed brain tissue, by use of 3 different antichlamydial antibodies. There was no staining with any of the 3 antichlamydial antibodies in formalin-fixed brain tissue from OND controls (n = 17). With available frozen CNS tissue, polymerase chain reaction (PCR) studies for the presence of C. pneumoniae genes were performed. The presence of a PCR signal was confirmed in 5 of 8 MS cases and in 3 of 18 OND controls. In an examination of CSF sediment by electron microscopy, we observed electron-dense structures resembling chlamydial organisms in CSF sediments from 11 of 20 MS cases and 2 of 12 OND controls. The presence of immunogold-labeled electron-dense bodies was correlated with the presence of a PCR signal in 10 of 11 MS cases. Results of studies using these different approaches support our suspicion of the presence of chlamydial organisms in the CNS, in a subset of patients with MS.
Chlamydia pneumoniae is an obligate intracellular microorganism that is a common cause of respiratory infections in humans [1, 2]. The ability of this organism to persist in infected tissue for extended periods has suggested that it may participate in either the development or the progression of chronic inflammatory disordersspecifically, atherosclerosis and reactive arthritis [36]. A number of studies have reported development of acute neurological syndromes after C. pneumoniae infection [715]. There is considerable debate regarding the role of C. pneumoniae persistence and infection in chronic diseases of the central nervous system (CNS). Nonetheless, a recent study has suggested that C. pneumoniae infection may be associated with Alzheimer disease, and we and others have suggested that C. pneumoniae infection may play a role in the pathogenesis of multiple sclerosis (MS) [10, 11, 16, 17].
Although the etiology of MS is not known, clinical observations suggest an interaction between an infectious agent and an autoimmune response to neural antigens in the development of the disease [18]. MS shows close similarities to autoimmune experimental allergic encephalitis, and epidemiologic studies have implicated infectious agents as a contributing element in the development of MS [19]. To reconcile the 2 hypotheses pertaining to autoimmunity and infection, it has been suggested that an infectious agent may act as a trigger in initiating an autoimmune process in the CNS [2023].
In the Nurses Health Study, antibody titers to C. pneumoniae were found to be higher in patients with progressive MS than in control individuals, suggesting that C. pneumoniae infection may be a factor in disease progression [24]. Our studies have shown that the major outer membrane protein (MOMP) DNA of C. pneumoniae is seen in the cerebrospinal fluid (CSF) from some patients with MS (hereafter referred to as "MS cases") but not in patients with other neurological diseases (hereafter referred to as "OND controls"). We and others have also noted the presence of antibodies to C. pneumoniae in CSF from some MS cases [16, 25]. Some investigators have confirmed the presence of C. pneumoniae DNA in CSF from MS cases [15, 19, 2630]; others have been unable to detect chlamydial DNA in MS cases or in control individuals [3133]. Technical differences in the methodologies of polymerase chain reaction (PCR) assays may account for the discrepancies.
One of the requirements for demonstrating a causal or influencing relationship between an infectious agent and a disease is demonstration of the presence of the organism in involved tissue. In the study presented here, we have examined CNS tissue and CSF sediment from MS cases and OND controls for evidence of the presence of C. pneumoniae.
PATIENTS, MATERIALS, AND METHODS
Patients.
With institutional review board approval, CSF was obtained from MS cases and OND controls. Postmortem brain tissue samples for immunohistochemical (IHC) staining and PCR analysis were obtained from archived specimens at the Vanderbilt Medical Center and from specimens available through the Rocky Mountain Brain Bank. In 11 cases, neither immunosuppressive nor immunomodulatory drugs were administered immediately before death. In the remaining 9 cases, pertinent clinical information on the use of immunosuppressive drugs during the period immediately before death was not available. In these cases, information on duration, progression, and severity of disease was also not available. The diagnosis and the number of cases examined for the control group were as follows: Parkinson disease (n = 4), Alzheimer disease (n = 3), systemic lupus erythematosis (n = 2), and 1 case each of subacute sclerosing panencephalitis, CNS lymphoma, adrenoleucodystrophy, cytomegalovirus encephalitis, and HIV encephalitis. Three patients1 who had died of bone marrow failure, 1 who had died of multiorgan failure after renal transplant, and 1 who had died of end-stage renal hypertensionwere also studied. In 5 MS cases and 4 OND controls, frozen tissue was available for PCR assays. Additional frozen tissue from 3 MS cases and 14 OND controls was obtained from archived material at the Vanderbilt Medical Center and from the Rocky Mountain Brain Bank for PCR assays. CSF from MS cases and OND controls was obtained as part of the diagnostic studies performed to evaluate patients seen in the MS clinic.
Reagents.
Antichlamydial antibodies were purchased from commercial sources and used at the concentrations shown in table 1. CF-2 antichlamydial lipopolysaccharide (LPS) antibody and antichlamydial anti-hsp60 antibodies have been recommended as optimal reagents for tissue staining [34, 35].
IHC staining of formalin-fixed CNS tissue.
Five-micrometer sections of formalin-fixed brain tissue was deparaffinized, blocked with peroxidase and casein, and incubated overnight with 1 of the 3 antichlamydial antibodies or their isotype-matched controls at dilutions shown in table 1. Control antibodies were used at the same concentrations as the antichlamydial antibodies. After overnight incubation, the sections were washed, and goat anti-mouse antibodies were added for 30 min at room temperature. The slides were washed, and the AEC protocol (Envison kit; DAKO) was used for the detection of binding to the primary antibody. The efficacy of the different antibodies was ascertained by staining mouse lungs infected with C. pneumoniae (mouse tissue blocks were a gift from T. Nagy, Department of Pathology, Vanderbilt Medical Center).
Immunogold staining of CSF sediment.
Twenty milliliters of CSF from each of 20 MS cases and 12 OND controls were spun at 65,000 g for 30 min, and the sediment was resuspended in 100 L of PBS. CSF sediments were placed on formvar-coated nickel grids and dried at 4°C. The grids were fixed in 4% paraformaldehyde-PBS for 15 min, washed and incubated with 50 mmol/L glycine for 10 min, and washed and blocked with 5% bovine serum albumin. Slides were incubated overnight with monoclonal antibody (MAb) 807 at a dilution of 1 : 50. After washing, a 1 : 50 dilution of gold-conjugated (10 nm gold; EM Sciences) anti-mouse antibody was added for 18 h. After washing, the grids were postfixed in 2% glutaraldehyde for 10 min, washed, air-dried, and examined by electron microscopy (EM).
Five randomly selected squares on each Formvar grid were examined, and electron-dense bodies bearing at least 15 gold spheres on the surface were considered to be positive. The total number of immunogold-stained cells in 5 squares was counted. The evaluator was blinded as to the diagnosis of each of the CSF specimens. We arbitrarily chose the presence of at least 4 positively stained electron-dense bodies on 5 squares in the formvar grids as a cutoff number for positive samples. Positive controls for the study included elementary bodies that were isolated from human lung cells infected with C. pneumoniae.
PCR of C. pneumoniae DNA from frozen brain tissue of MS cases and OND controls.
Frozen tissue stored at -70°C was thawed to -20°C and dissected in a sterile hood. C. pneumoniae is a ubiquitous organism, and, therefore, DNA isolation was performed using aseptic techniques [36]. Where possible, subependymal (periventricular) lesions were identified and carefully dissected for PCR assays. Tissue (2.5 mg) was homogenized and lysed in 500 L of PCR lysis buffer; 25 L of proteinase K was then added, and the mixture was incubated overnight at 37°C. DNA was extracted from the tissue by use of the chloroform/phenol method [12].
Nested PCR studies were performed using primers specific for the C. pneumoniae MOMP and 16s RNA genes. The primers for the MOMP gene were as follows: external, sense (5-AAC TAT ACT ACT GCC GTA GA-3) and antisense (5-GTA GTA GAC AAT GCT GTG G-3); and nested, sense (5-ACA CCT CTT TCT CTT GGA GCG T-3) and antisense (5-TTG ATG GTC GCA GAC TTT GTT C-3). The primers for the 16s RNA gene were as follows: external, sense (5-GCT AAT ACC GAA TGT AGT GTA A-3) and antisense (5-ATC TAT CCT CTA GAA AGA TAG TT-3); and nested, sense (5-GTA AAA GCC CAC CAA GGC GAT GA-3) and antisense (5-CTA CAC GCC CTT TAC GCC CAA-3).
The extracted DNA (5 L) in extraction buffer was added to 10× PCR buffer, 1.5 mmol/L MgCl2, 200 mol/L dNTP, 25 pmol of each primer, and 1 U of AmpliTaq polymerase (Roche), in a volume of 25 L. DNA amplification was performed using a touchdown/nested PCR technique. After a preheating cycle at 94°C for 5 min, amplification of the outside product was performed first by use of a touchdown technique in which the annealing temperature was reduced 0.5°C every cycle, from 58°C to 48°C, and further amplification was performed for 30 cycles at 48°C, followed by an extension of the product at 72°C for 5 min. For amplification of 16s ribosomal DNA, the annealing temperature during the touchdown phase was decreased from 60°C to 50°C. The nested amplification consisted of a denaturation step at 94°C for 45 s, an annealing stage for 45 s (58°C for MOMP and 60°C for 16s ribosomal DNA, respectively), and an extension phase at 72°C for 1 min. The PCR product was run on a 1.5% agarose gel and stained with ethidium bromide. CSF or water spiked with 0.4 inclusion-forming units (IFUs) of C. pneumoniae elementary bodies was used as a positive internal control.
Ultrastructural examination of CSF sediments.
CSF sediments were obtained from 20 mL of CSF from each of 10 MS cases and 5 OND controls. Each sample was centrifuged at 30,000 g for 15 min, and each "button" of sediment was fixed directly in its centrifuge tube in 1.25% glutaraldehyde for 2 h, postfixed in 1% osmium for 1 h, dehydrated, and embedded in Spurr's Embedding Medium (Polysciences). Ultrathin (500 ) sections were placed on 300-mesh copper grids, stained with lead acetate stain, and examined by EM.
RESULTS
IHC staining of chlamydial antigens in the CNS.
We examined 5-m sections of 30 paraffin blocks from 20 MS cases and 25 blocks from 17 OND controls, using IHC techniques for identifying the presence of C. pneumoniae antigens (a representative selection of cases for which both PCR and IHC techniques were performed is shown in tables 2 and 3). Staining with all 3 antichlamydial antibodies and the respective control antibodies was performed. The criterion used to conclude that a particular section was positive was confirmation of antibody staining with all 3 of the antichlamydial antibodies (figures 1A and 2). The specificity of staining was established by performing IHC studies on mouse lungs infected with C. pneumoniae (figure 1B). Among the 20 MS cases, 7 showed staining with all 3 antichlamydial antibodies. Staining was repeated at least 3 times, and similar results were obtained. In 1 MS case, prominent staining for MAb 807 was seen in a tuft of ependyma, but staining with CF-2 and anti-hsp60 antibodies was not performed. In the sections showing positive staining, the antibody staining was seen in some but not all ependymal cells of the ventricular wall. In the positively staining cells, the staining showed a granular pattern (figures 1 and 2). In some sections, the staining appeared to be intranuclear (figure 2), but, since chlamydial antigens are not known to be present in the nucleus, we interpret these findings as indicating that the perinuclear location of chlamydial antigens gives a false appearance of intranuclear localization. Along with staining of ependymal cells, staining was also seen in glial cells and perivascular cells in the subependymal layer (figures 3 and 4). Although many of the parenchymal regions of the white matter showed presence of an active inflammatory response, with rare exception these regions lacked staining with any of the antichlamydial antibodies (figure 5). In the OND controls, we did not detect staining of CNS tissue with any of the antichlamydial antibodies.
Identification of C. pneumoniae DNA by PCR in frozen CNS tissue of MS cases and OND controls.
Frozen tissue from 8 MS cases and 18 OND controls were available for analysis. Of these, 5 MS cases and 4 OND controls were also examined by IHC staining (tables 2 and 3). Nested PCR was performed using 2 different primers. Amplification of MOMP and 16s RNA DNA was possible with the frozen tissue from 5 of 8 MS cases. Three of these cases were also positive for chlamydial antigens by IHC staining (P < .05, vs. OND controls). MS case 2 (table 2) was positive by IHC staining, but a PCR signal was detected only with MOMP primers. For MS case 8 (table 2), tissue was positive by PCR, but formalin-fixed tissue was not available for IHC staining. We also studied 18 OND controls in a similar manner. These included OND controls 14 (table 3), for whom IHC staining with antichlamydial antibodies were performed.
Of the 18 OND controls studied, 3 were positive by PCR with both primers (table 3). These included 1 with Alzheimer disease, another with multisystem atrophy, and a third with Devic disease. Three controls1 with amyotrophic lateral sclerosis, another with a diagnosis of Parkinson disease and Alzheimer disease, and a third with bone marrow failurewere positive by PCR after amplification of the MOMP gene alone. Interestingly, 1 of the controls with Alzheimer disease was positive by PCR in the cerebellum.
Immunogold staining of CSF sediment with antichlamydial antibodies and the detection of a PCR signal for C. pneumoniae DNA in CSF.
Our observation of chlamydial antigens in close proximity to the ventricular system and the presence of C. pneumoniae DNA in CSF from MS cases suggested that a direct examination of chlamydial elementary bodies in CSF may be productive. Eleven of 20 MS cases had 4 cells in 5 squares stained with MAb 807. In contrast, CSF sediment from only 2 of 12 OND controls was positive. One of these 2 had a diagnosis of acute disseminated encephalomyelitis (ADEM), and the other had an as-yet-undiagnosed CNS disease, with abnormal lesions found on magnetic resonance imaging in the CNS white matter (P < .025, vs. OND controls) (figure 6). Many of the electron-dense bodies showed a morphology that was either oval or pear shaped, similar to that seen in the positive controls (figure 7).
We next explored whether the immunogold staining for C. pneumoniae in CSF sediment was correlated with a PCR assay for the presence of C. pneumoniae MOMP DNA obtained from the same aliquot of CSF (table 4). Of the 20 MS cases, 12 (60%) were positive by PCR for the C. pneumoniae MOMP gene. One (MS case 12; table 4) did not show IHC staining of CSF sediment but was PCR positive for MOMP DNA. Of the OND controls, 1 with ADEM (OND control 5; table 4) was found by PCR to be positive for the C. pneumoniae MOMP gene, and the remaining 11 were negative (P < .02, MS cases vs. OND controls). These studies provide evidence that immunogold-stained electron-dense bodies resembling elementary bodies of C. pneumoniae can be detected in CSF from MS cases with greater frequency than in CSF from OND controls, and these observations were correlated with the results of accompanying PCR studies performed with CSF obtained at the same time.
Direct EM study of CSF sediment from MS cases.
CSF sediment from another set of 10 MS cases and 5 OND controls was examined ultrastructurally. Structures resembling chlamydial bodies were found to be present in samples of CSF sediment from 4 MS cases but not in those from any of the OND controls (figure 8). The diameter of these bodies ranged from 0.25 to 0.70 m, and the outer membrane showed a trilaminar organization. Furthermore, the surfaces of the outer membrane had knoblike projections that were similar to those described in scanning EM studies of other chlamydial species (figure 8) [3739]. These bodies were not found in any of the CSF sediment from OND controls.
DISCUSSION
The studies reported here show evidence of the presence of chlamydial antigens in postmortem brain tissue samples from MS cases. Structures that resemble elementary bodies of chlamydial organisms were observed in CSF from MS cases, and IHC studies demonstrated localization of the chlamydial antigens in the ventricular wall and subependymal regions in MS cases but not in OND controls. Parallel studies performed using PCR techniques with CNS tissue showed that the C. pneumoniae DNA was found in the same regions. CSF analysis showed the presence of C. pneumoniae MOMP DNA in CSF from MS cases more commonly than in CSF from OND controls. It would have been ideal to demonstrate the presence of chlamydial organisms in the CNS by all available methods. However, attempts to demonstrate the presence of chlamydial DNA by use of in situ hybridization and PCR assays on formalin-fixed brain tissue were technically unsuccessful.
IHC staining of paraffin-fixed postmortem brain tissue from MS cases demonstrated specificity as indicated by the similarity in quality and localization of staining with all 3 antibodies (MAb 807, CF-2, and anti-hsp60). The morphological characteristics of the staining were also similar to those published elsewhere showing the presence of C. pneumoniae in vascular tissue [3, 4042]. These antibodies recognize chlamydial antigens that are genus specific but not species specific and are therefore not specific for C. pneumoniae; however, since the accompanying PCR studies were performed with primers specific for C. pneumoniae, it is likely that the antigens found in the brains are those of C. pneumoniae. Although monoclonal antibodies RR402 and TT401, which are specific for C. pneumoniae, are available, IHC staining with these antibodies was inconsistent and required high concentrations (dilutions of 1 : 2 or 1 : 5). Although other studies have shown the presence of C. pneumoniae antigens in the brains of patients with Alzheimer disease, we did not see staining with our panel of antibodies in brain tissue from the patient with Alzheimer disease included in the present study [10]. However, we performed IHC staining in the periventricular regions but not in the gray matter, and that might account for the discrepancy.
Since we were not able to extract nondegraded DNA from paraffin-embedded tissue, DNA for PCR analysis was obtained from frozen brain tissue in periventricular regions that had been demonstrated to be positive for chlamydial antigens by IHC staining. Although C. pneumoniae was not exclusively present in MS brain tissue, it was identified in more MS cases than OND controls. The morphological characteristics of the bodies identified by ultrastructural examination of CSF in this study resemble structures of Chlamydia species. These bodies have a bilaminar membrane and show arrays of surface projections that are virtually identical in appearance to those that have been observed in C. trachomatis and C. psittacci [37, 39].
It is important to point out that we did not identify any IHC staining in regions of active inflammation. This is an intriguing and puzzling observation, since we had anticipated that antigenic evidence of the pathogen's presence would be seen in close proximity to inflammation. We assumed that migration of monocytes infected with C. pneumoniae would represent a conduit for its entry into the CNS and, hence, that its presence would be seen in perivascular regions. In some diseases of the CNS that are well recognized as being infectious, the route of entry of the pathogen is not always clear [43, 44]. The IHC staining for chlamydial antigens in the ependyma and in the subependymal regions may suggest an alternate route of entry. We suggest that the circumventricular organs may provide a route of entry for infected mononuclear cells into the CNS. These regions contain loosely structured vascular tissue and compose the area postrema: the caudal region of the fourth ventricle, median eminence, subcomissural organ, and subfornicular organ [45, 46]. They all lie close to the ventricular space, and the capillary blood vessels in these regions lack tight junctions and astrocytic foot processes and, hence, do not exhibit the restrictions in trafficking imposed by the blood-brain barrier. Also, the overlying ependyma lack cilia and resemble subependymal glial cells. Studies have shown that, although these regions are small in area, they are sufficient to allow for the migration of compounds that would otherwise be restricted to the vascular compartment [47].
Ependymal cells in MS cases sometimes show features of inflammatory injury. In a series of 129 cases, Adams noted active ependymitis in 11% of MS periventricular lesions, characterized by ependymal granulations [48]. In other regions of the ventricle, chronic ependymal reaction was seen, including flattened ependymal cells and subependymal gliotic nodules [49]. It is therefore conceivable that ependymal and circumventricular organs may be the site of involvement in CNS inflammation, most notably that associated with MS. The absence of the organism in parenchymal regions of active inflammation suggests that infection of CNS tissue may be an epiphenomenon. However, there are a number of chronic diseases in which inflammation is seen in the absence of an abundance of pathogen. In tuberculoid leprosy, granuloma formations are seen in regions in which the bacilli are either absent or few in number [50]. Paucity of organisms is also seen in CNS syphilis and CNS Lyme disease. The inflammatory response may have more to do with the nature of the host response to the inciting pathogen than with the number or the proximity of the pathogen to the areas of tissue injury [51, 52].
The studies presented here, along with our previous PCR studies of the presence of C. pneumoniae DNA in CSF from a subset of MS cases, suggest an association between this pathogen and disease. These studies do not provide evidence of a causal role between C. pneumoniae infection and MS; however, chlamydial antigens may be an infectious "trigger" that initiates or leads to autoimmunity. Molecular mimicry between C. pneumoniae antigens and myelin antigens has been recognized; immunization of animals with these cross-reactive antigens is sufficient to induce autoimmune disease [53, 54]. Alternatively, the infection may act as a secondary invader in host tissue, accentuating an ongoing inflammatory response [55].
Acknowledgments
We thank Anthony Tharp for technical assistance with the electron microscopy studies. We also thank Camilla Dietz Bergeron, Thomas West, Paul Griffin, Steve Smith, and the family of C. J. Schueler, for their support.
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