New insights into neuropathology and pathogenesis of autoimmune glial fibrillary acidic protein meningoencephalomyelitis

Abstract

Anti-glial fibrillary acidic protein (GFAP) meningoencephalomyelitis (autoimmune GFAP astrocytopathy) is a new autoimmune central nervous system (CNS) disease diagnosable by the presence of anti-GFAP autoantibodies in the cerebrospinal fluid and presents as meningoencephalomyelitis in the majority of patients. Only few neuropathological reports are available and little is known about the pathogenic mechanisms. We performed a histopathological study of two autopsies and nine CNS biopsies of patients with anti-GFAP autoantibodies and found predominantly a lymphocytic and in one autopsy case a granulomatous inflammatory phenotype. Inflammatory infiltrates were composed of B and T cells, including tissue-resident memory T cells. Although obvious astrocytic damage was absent in the GFAP-staining, we found cytotoxic T cell-mediated reactions reflected by the presence of CD8⁺/perforin⁺/granzyme A/B⁺ cells, polarized towards astrocytes. MHC-class-I was upregulated in reactive astrocytes of all biopsies and two autopsies but not in healthy controls. Importantly, we observed a prominent immunoreactivity of astrocytes with the complement factor C4d. Finally, we provided insight into an early phase of GFAP autoimmunity in an autopsy of a pug dog encephalitis that was characterized by marked meningoencephalitis with selective astrocytic damage with loss of GFAP and AQP4 in the lesions.Our histopathological findings indicate that a cytotoxic T cell-mediated immune reaction is present in GFAP autoimmunity. Complement C4d deposition on astrocytes could either represent the cause or consequence of astrocytic reactivity. Selective astrocytic damage is prominent in the early phase of GFAP autoimmunity in a canine autopsy case, but mild or absent in subacute and chronic stages in human disease, probably due to the high regeneration potential of astrocytes. The lymphocytic and granulomatous phenotypes might reflect different stages of lesion development or patient-specific modifications of the immune response. Future studies will be necessary to investigate possible implications of pathological subtypes for clinical disease course and therapeutic strategies.

Keywords: Autoimmunity; Autopsies; Biopsies; GFAP; Magnetic resonance imaging.

Fig. 1

Neuropathological findings of anti-GFAP meningoencephalitis – autopsy case #1 The inflammatory reaction is characterized by perivascular cuffs with abundant CD3⁺ (a), CD4⁺ (b) and CD8⁺ T cells (c), as well as CD20⁺ B cells (d). In the brain parenchyma, CD3⁺ (e) and CD8⁺ T cells (g) were more abundant compared to CD4⁺ (f) and CD20⁺ (h) lymphocytic infiltrates. Reactive gliosis in the depth of the sulci (i, asterisks, GFAP) is present subjacent to meningeal lymphocytic infiltrates (i, arrowheads, GFAP). AQP4 is upregulated and mainly found in subpial astrocytes (j, arrowheads). C4d indicates complement deposition on astrocytes (k, arrow in k enlarged in panel l), confirmed with double labeling for C4d (blue) and GFAP (red) (m). MHC class I molecules are upregulated in some astrocytes (n). Deposition of C4d on NRP1-positive glial cells is confirmed with the double labeling for NRP1 (blue) and C4d (red) (o). Scale bars a–d, i, j 100 µm; e–h 50 µm; k 500 µm; l-o 25 µm. AQP4 aquaporin 4, CD cluster of differentiation, C4d complement split product 4d, GFAP glial fibrillary acidic protein, MHC-I major histocompatibility complex class I, NRP1 neuropilin 1

Fig. 2

Topographical mapping and imaging characteristics of inflammatory infiltrates of autopsy case #1 Topographical evaluation of a double-hemispheric brain section shows a widespread distribution of CD20⁺ B cells (a; red dots represent immunolabeled cells) in the meninges (a, green arrow enlarged in panel b), in perivascular cuffs (a, orange arrow enlarged in panel d) and in the brain parenchyma. Corresponding in vivo brain MRI shows multiple areas of gadolinium enhancement in post-contrast T1 images (c, green arrow indicates meningeal inflammation, orange arrow indicates the corresponding place where abundant number of B lymphocytes were present around blood vessels). Anatomic distribution of inflammation in a more anterior brain section including the basal ganglia similarly shows a maximum of CD20⁺ B cells mainly around the ventricles (e; red dots represent immunolabeled cells). Colored arrows in e and h (enlarged in f, g, i, j) indicate a profound number of perivascular CD20⁺ B cells that are visible as gadolinium enhancement in the corresponding in vivo MR image (h). Scale bars b, d 500 µm; inset in b and d 50 µm; f, g, i, j 25 µm

Fig. 3

Magnetic resonance imaging findings of anti-GFAP meningoencephalomyelitis – autopsy case #2 Brain in vivo MRI from autopsy patient #2 shows a bilateral optic neuritis (a, white arrows) on post-contrast axial angulated reconstructions of T1-weighted blackblood-images. Further axial angulated reconstruction of post-contrast T1-weighted blackblood-images shows enhancement throughout the cisternal segment of both trigeminal nerves (b). The right trigeminal nerve additionally shows an abnormal enhancement in the trigeminal cave (b, white arrow), indicating an involvement of the ganglion

Fig. 4

Neuropathological findings of anti-GFAP meningoencephalomyelitis – autopsy case #2 In the chiasma opticum numerous CD3⁺ (a), CD4⁺ (b) and CD8⁺ T cells (c) and CD20⁺ B cells (d) are present in the leptomeninges and around vessels. Parenchymal CD8⁺ T cells (red) are found in close proximity to GFAP positive astrocytes (blue) (e). Some of these T cells express granzyme A (red) (f). The perivascular granulomas contain multinucleated giant cells of the Langhans type (arrow in g enlarged in inset h), and a high number of CD3⁺ (i), CD4⁺ (j) and CD8⁺ T cells (k), and less CD20⁺ B cells (l). pSTAT1, a marker for interferon signaling, is strongly upregulated in nuclei of multinucleated giant cells (m) and CD4⁺ (n) and CD8⁺ T lymphocytes (o). CD103⁺ tissue resident memory T cells are present in the granulomas as well as in the brain parenchyma (p). In the cerebellum, granulomas are evident in the cortex with prominent astrogliosis (arrow in q enlarged in inset r) with well-preserved astrocytic processes (r) and microglia activation (arrow in s enlarged in inset t). The proliferation marker Ki-67 (green) is expressed in the nucleus (blue; DAPI) of some astrocytes (red; GFAP) (u). Scale bars a-d, q, s 250 µm; e, f, n, o 6 µm; g, i-m 50 µm; h, p, r, t 10 µm; u = 5 µm. AQP4 aquaporin 4, CD cluster of differentiation, DAPI 4’,6-diamidino-2-phenylindole, GFAP glial fibrillary acidic protein, GzA granzyme A, H&E hematoxylin & eosin, HLA human leukocyte antigen, pSTAT1 phosphorylated signal transducer and activator of transcription 1

Fig. 5

Inflammatory infiltrates in the posterior spinal roots of autopsy case #2 In the spinal roots, CD3⁺ (a), CD4⁺ (b), CD8⁺ (c), and CD20⁺ (d) lymphocytes are predominantly found in the peri- and epineurium, in addition scattered lymphocytes are found in the endoneurium. Scale bars a-d 100 µm. CD cluster of differentiation

Fig. 6

Inflammation involving both CNS gray and white matter in GFAP autoimmunity (a-d; meninges and cortex, biopsy findings) Consecutive sections showing meningeal and gray matter inflammation in the brain: (a) H&E stain reveals hypercellularity in both meninges and subpial cortex. (b) Marked microglial activation. (c) T cell infiltration in both meninges and cortex. (d) B lymphocytes remain within the meninges, without cortical parenchymal infiltration. (e–g; white matter) Consecutive sections show hypercellularity (e, H&E), microglial activation (f) and CD8⁺ T cell (g) parenchymal infiltration in the white matter of the brain. (h, inset enlarged) Eosinophilic granulocyte infiltration is occasionally seen in the perivascular region. (i-l; parenchyma) Consecutive sections show diffuse and perivascular inflammation in the brain parenchyma: (i) T lymphocytes are present in both parenchyma and perivascular region. (j) B lymphocytes are limited to the perivascular space. (k) A minority of T lymphocytes are positive for CD4. (l) Most T lymphocytes are CD8 positive. (m-t; microglial nodules) Consecutive sections showing microglial nodules: (m) Iba-1 stain indicates microglial nodule formation. (n) CD8-positive T lymphocytes are present in the nodule. (o) No B lymphocytes are present within the nodule. (p) Perforin labels some T lymphocytes. (q) Granzyme A (GzA) positive stain indicates cytotoxic T cells, magnified in (r). (s) Granzyme B (GzB)-positive cells are absent within this microglial nodule. (t) The negative control by omitting the primary antibody indicates the specificity of the immunohistochemistry system. Single biopsy shows mixed lymphocytic and granulomatous patterns of inflammation (u-x): (u) One biopsy harbors a multinucleated giant cell (indicated with arrows) present in the perivascular regions with lymphocytic infiltration (H&E). (v) Immunohistochemistry on consecutive section reveals the expression of CD68, a phagocytosis marker of myeloid cells, in the multinucleated giant cells (pointed with arrows). (w) A different location in the same biopsy shows perivascular and diffuse T cell infiltration (CD3). (x) A major proportion of these T cells shows CD8 positivity. Scale bars a-d 100 µm; e–g, i-t, w, × 50 µm; h, u, v 20 µm. CD cluster of differentiation, GzA granzyme A, GzB granzyme B, H&E hematoxylin & eosin, Iba-1 ionized calcium-binding adapter molecule 1, Neg negative control

Fig. 7

MHC-1 expression in reactive astrocytes and cytotoxic T lymphocytes is present in the CNS of GFAP autoimmunity (a) The normal CNS shows limited expression of MHC-1 in the endothelial cells, which is not detectable in astrocytes. (b) The brain biopsy of a patient with anti-GFAP autoantibodies shows increased MHC-1 immunoreactivity in both cytoplasm and cell processes. Note some infiltrating leukocytes show strong membrane MHC-1 immunoreactivities. (c) The magnified view of (b) shows increased MHC-1 signals in cytoplasm and processes in a hypertrophic reactive astrocyte. (d-f) and magnified views (g-i) show cytotoxic T lymphocyte markers on consecutive tissue sections: more T lymphocytes show granzyme A (GzA) positivity (d, g) than granzyme B (GzB) (e, h). Perforin-positive cells (f, i) are numerically similar to granzyme A. (j) Double immunostain shows a fraction of lymphocytes positive for CD8 (red) and granzyme A (GzA, brown). (k) Many CD8-positive T cells (brown) are also positive for CD103 (red). The left inset shows a magnified view of a CD8⁺CD103⁺ cell. The right inset shows a CD8⁻CD103⁺ cell. (l) A few CD4 lymphocytes are also positive for CD103. The left inset shows a CD4⁺CD103⁺ cell. The right inset shows a CD4⁺CD103⁻ cell. (m) GFAP stain shows diffuse hypertrophic astrocytes around an inflamed vessel. (n) Double immunostain for CD8 (brown) and GFAP (red) indicates the close contact between astrocytes and CD8⁺ lymphocytes. (o) Double stains for granzyme A (GzA, brown) and GFAP (red) show a cytotoxic T lymphocyte polarizing the granzyme A granules towards the astrocyte process (inset). Scale bars a, b, g-l, n, o 20 µm; c 3 µm; d-f, m 50 µm. CD cluster of differentiation, GFAP glial fibrillary acidic protein, GzA granzyme A, GzB granzyme B, MHC-1 major histocompatibility complex class I

Fig. 8

Subpial pathology and parenchymal aquaporin loss in GFAP autoimmunity (a) H&E staining shows hypercellularity in the leptomeninges and subpial parenchyma. Increased collagen is noted in the leptomeninges (magnified view in panel d). (b) CD68 on the consecutive section highlights marked macrophages/microglia in both meninges and subpial parenchyma (high power view in panel e). (c) Infiltration of T lymphocytes in meninges and towards the deep parenchyma is seen in the same region (magnified view in panel f). (g-j) GFAP staining indicates alteration of glia limitans in the inflamed area. (h) Decreased GFAP along pial surface. (i) The high-power view highlights discontinuous glia limitans and some adjacent hypertrophic astrocytes. (j) The adjacent non-inflamed region shows intact glia limitans. (k) Immunohistochemistry indicates decreased pial AQP4 immunoreactivity in the highly inflamed region (high power view in l). (m) Decreased pial AQP1 is also noted. (n) Cortical perivascular loss of AQP1 immunoreactivity. (o) Perivascular decreased AQP4 immunoreactivity. Scale bars a-c 100 µm; g, m 200 µm; l, n, o 50 µm; h, k 100 µm; d-f, i, j 20 µm. AQP1 aquaporin 1, AQP4 aquaporin 4, CD cluster of differentiation, GFAP glial fibrillary acidic protein, H&E hematoxylin & eosin

Fig. 9

Neuropathological findings in a canine autopsy case with anti-GFAP autoantibodies Overview stainings (H&E (a); AQP4 (b); GFAP (c)) of the same area of a canine temporal brain section including the hippocampus. H&E staining (d) shows edema, reactive gliosis, neutrophilic granulocytes and mitoses and perivascular inflammatory infiltrates mainly consist of lymphocytes and plasma cells (arrow in d enlarged in inset e). In some parts of the lesions AQP4 (f) and GFAP (g, h) are selectively lost (h) with a few apoptotic astrocytes remaining within the lesion (i), while axons are relatively well preserved (j; Biel). Other parts of the lesions showed focal cystic necrosis with abundant CD68 positive macrophages (k) and loss of axons (l; Biel). Immunohistochemistry reveals some CD20⁺ B cells (m) around vessels. Inflammation is characterized by an abundant number of perivascular and parenchymal CD3⁺ T cells (n). pSTAT1 is mainly upregulated in lymphocytes (o). Scale bars d, m, n 50 µm; f, g 500 µm; e, i, o 10 µm; h, j-l 25 µm. AQP4 aquaporin 4, Biel Bielschowsky, CD cluster of differentiation, GFAP glial fibrillary acidic protein, H&E hematoxylin & eosin, pSTAT1 phosphorylated signal transducer and activator of transcription 1