Shuji Izumo, MD, PhD, Molecular Pathology, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8544, Japan. Email: firstname.lastname@example.org
A series of our neuropathological studies was reviewed in order to clarify pathogenesis of human T lymphotropic virus type 1(HTLV-1)-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). The essential histopathologic finding was chronic inflammation in which inflammatory infiltrates of mononuclear cells and degeneration of myelin and axons were noted in the entire spinal cord. Immunohistochemical analysis demonstrated T-cell dominance, and the numbers of CD4+ cells and CD8+ cells were equally present in patients with shorter clinical courses. Apoptosis of helper/inducer T-cells were observed in these active inflammatory lesions. Horizontal distribution of inflammatory lesions was symmetric at all spinal levels and was accentuated at sites with slow blood flow in the middle to lower thoracic levels. HTLV-1 proviral DNA amounts were well correlated with the numbers of infiltrated CD4+ cells. In situ PCR of HTLV-1 proviral DNA and in situ hybridization of HTLV-1 Tax gene demonstrated the presence of HTLV-1-infected cells exclusively in the mononuclear infiltrates of perivascular areas. From these findings, it is suggested that T-cell mediated chronic inflammatory processes targeting the HTLV-1 infected T-cells is the primary pathogenic mechanism of HAM/TSP. Anatomically determined hemodynamic conditions may contribute to the localization of infected T-cells and the formation of main lesions in the middle to lower thoracic spinal cord.
Human T lymphotropic virus type 1 (HTLV-1) is the first recognized human retrovirus and is found to be a causative agent of adult T-cell leukemia/lymphoma (ATL).1 Epidemiological survey of ATL and HTLV-1 seropositive carriers demonstrated the deviated distribution to southwestern Japan. In 1985, Osame and colleges noticed in one of the most endemic areas of HTLV-1, Kagoshima, that some patients manifesting slowly progressive spastic paraparesis with sphincter dysfunction had antibodies against HTLV-1 in both their sera and CSF. Further analysis of anti-HTLV-1 antibodies on stored CSF specimens from various neurological diseases found additional cases with slowly progressive spastic paraparesis having anti-HTLV-1 antibodies. Their hematological features did not satisfy diagnostic criteria of ATL. Based on these finding, the term HTLV-1-associated myelopathy (HAM) was proposed as a new clinical entity.2 Independently, Gessain et al. have reported that about 60% of Caribbean patients with tropical spastic paraparesis (TSP) were seropositive for HTLV-1.3 HAM and HTLV-1-positive TSP were later confirmed as a single clinical entity and the name HAM/TSP was recommended by WHO.
CLINICAL FEATURES AND LABORATORY FINDINGS OF HAM/TSP4
HAM/TSP is characterized by a spastic paraparesis with urinary disturbances and anti-HTLV-1 antibody positivity in serum and CSF. Almost all patients show spasticity and/or hyper-reflexia of the lower extremities. Many patients manifest weakness of the lower extremities and a poorly defined (mild) sensory effect. These symptoms are generally slowly progressive, or in some cases static after initial progression, while patients at older ages of onset show faster progression regardless of the mode of transmission.
Patients with HAM/TSP have high antibody titers to HTLV-1 both in serum and CSF. Aside from HTLV-1 antibody positivity, other essential laboratory findings include lymphocytic pleocytosis in the CSF and increased CSF neopterin levels. In MRI, high signals on T2-weighted images are observed in the white matter of the brain similar to those found in multiple sclerosis. Swelling or atrophy of the spinal cord has been reported in some cases of HAM/TSP.
NEUROPATHOLOGY AND PATHOGENESIS OF HAM/TSP
The first autopsy case of HAM/TSP was reported by Akizuki et al.,5 in which marked inflammatory infiltrates and diffuse loss of myelin and axons in the spinal cord were described as a histopathologic findings. Thereafter, more than 30 cases of autopsy have been reported, and most of them showed quite similar histopathologic findings.6,7
General neuropathologic findings
Macroscopically, the spinal cord shows symmetrical atrophy especially in the entire thoracic cord according to their severity of neurological deficits. Infiltration of mononuclear cells and degeneration of both myelin and axons are the essential microscopical findings of cases with relatively short clinical course of the disease (Figs 1,2). Inflammatory lesions are most severe in the middle to lower thoracic spinal cords and are continuously extended to the entire spinal cord. Similar but much milder lesions are scattered in the brain. On the other hand, in patients with prolonged clinical history, the spinal cord shows monotonous degeneration and gliosis with a few inflammatory cells in the perivascular areas. Fibrous thickening of the vessel walls and pia mater is frequently noted. These findings suggest a preceded inflammatory process in such areas. Degeneration of the spinal cord white matter is symmetric and diffuse but more severe at the anterio-lateral column and inner portion of the posterior column where the inflammatory lesions are accentuated in the active-chronic phase. Wallerian type fascicular degeneration is superimposed. There is no focal demyelinating plaque. Remaining myelinated fibers are randomly distributed in the diffusely degenerated lateral column. Inflammatory infiltrates and gliosis are also observed in the spinal cord gray mater. However, neuronal cells are relatively preserved.
Subsets of inflammatory cells, expression of cytokines and adhesion molecules, and apoptosis of T-cells in the spinal cord lesions
In the patients with shorter duration of illness, CD4+ cells, CD8+ cells and macrophages were evenly distributed in active inflammatory lesions. On the other hand, there is predominance of CD8+ cells over CD4+ cells in the inactive-chronic lesions of patients with longer duration of illness. Natural killer cells, IL-2 receptor positive cells and B-cells were only rarely present in both active and inactive inflammatory lesions.8 Cytokines such as IL-1β, tumor necrosis factor-α, and interferon-γ were expressed by macrophages, astrocytes, and microglia in the active inflammatory lesions.9 Among various adhesion molecules, spinal cord lesions of HAM/TSP have greater vascular cell adhesion molecule (VCAN)-1 expression on endothelium compared with those of controls, and infiltrating mononuclear cells expressed very late antigen (VLA)-4 especially in the perivascular lesions.10 These findings suggest that immune responses, especially T-cell mediated immune responses, take an important role in the spinal cord lesions of HAM/TSP.
We applied a monoclonal antibody T-cell restricted intracellular antigen (TIA)-1, a maker of cytotoxic T-cells, to detect possible effector cells of the inflammation in HAM/TSP. Many TIA-1+/CD8+ cells were distributed in the active inflammatory lesions; however, few cells were positive in the inactive chronic lesions. Because the protein TIA-1 has been reported in association with the induction of apoptosis in target cells, we carefully observed and found some cells undergoing apoptosis, most of them identified as CD45RO+ helper/inducer T-cells which are known as HTLV-1-harboring cells in vivo.11 These findings suggest that cytotoxic T-cell-mediated apoptosis of helper/inducer T-cells may be induced in the spinal cord of HAM/TSP patients.
Detection of HTLV-1 provirus in spinal cord lesions
It is crucially important to know whether there are HTLV-1-infected cells in inflamed spinal cord lesions. HTLV-1 proviral DNA could be detected in extracted DNA from affected spinal cord in HAM/TSP by PCR. The amount tended to decrease with the disease duration and this decline was paralleled with the decrease of CD4+ T-cell numbers.12 Based on these findings we applied PCR in situ hybridization (PCR-ISH) to determine which cells harbor the HTLV-1 provirus in vivo in the spinal lesions of HAM/TSP. Fresh frozen sections of the spinal cord were first immunostained with antibodies to T-cells and macrophages as well as helper/inducer T-cells, then PCR-ISH was carried out with specific primers and probed for the HTLV-1 pX region. PCR-ISH positive cells were exclusively detected among the T-cells around perivascular areas (Fig. 3) and about 10% of infiltrated T-cells were PCR-ISH positive in active-chronic lesions.13 Expression of Tax mRNA was also detected in the infiltrated T-cells of perivascular areas.14 These data are direct demonstrations of HTLV-1 infection to infiltrated T-cells in the spinal cord lesions. T cell-mediated immune responses targeting these infected cells may be a main event occurring in the spinal cord of HAM/TSP patients.
Inflammatory lesions are accentuated at sites with slow blood flow
It may be reasonable to suggest that the immune responses to HTLV-1 infected cells occur in the spinal cord of HAM/TSP because high immune responsiveness to HTLV-1 has been reported in HAM/TSP. However, why do such immune responses occur preferentially in the spinal cord, especially in the middle to lower thoracic level? To understand this point, we carefully analyzed distribution of inflammatory lesions in the entire CNS.15
In the spinal cord, inflamed vessels were symmetrically distributed and accentuated in the lateral column and the ventral portion of the posterior column, especially the middle to lower thoracic level. This distribution matches with the ending area of both the central and peripheral spinal arteries (Fig. 4). In addition, the anterior spinal artery of the middle to lower thoracic level has the most distant blood supply from the main trunk of the arteries, the vertebral artery and the Adam-Kiewicz artery, from the opposite directions, and this makes blood flow slower in that area. Inflammatory infiltrates in the brain were also distributed in the areas where there is slower blood flow.
Interestingly it has been reported that VCAM-1 was expressed on endothelial cells according to the decreased shear stress of blood flow. Further, expression of VCAM-1 and VLA-4 was increased in active-chronic lesions of HAM/TSP. We have also reported characteristic expression of matrix metalloproteinases16 and a novel variant of CD4417 in such active-chronic lesions. Using these molecules, HTLV-1-infected T-cells migrate into the CNS from the area where the blood flow is slow and initiate inflammatory lesions.
HAM/TSP is now a well-defined clinicopahological entity in which the virus infection and the host immune responses are involved in the pathogenesis. Our series of studies mentioned here suggested that T cell-mediated chronic inflammatory processes targeting the HTLV-1 infected T-cells are the primary pathogenic mechanism of HAM/TSP (Fig. 5).18 Anatomically determined hemodynamic conditions may contribute to the localization of infected T-cells and forming of main lesions in the middle to lower thoracic spinal cord.