Immune surveillance in the injured nervous system: T-lymphocytes invade the axotomized mouse facial motor nucleus and aggregate around sites of neuronal degeneration

Abstract

Although the CNS is an established immune-privileged site, it is under surveillance by the immune system, particularly under pathological conditions. In the current study we examined the lymphocyte infiltration, a key component of this neuroimmune surveillance, into the axotomized facial motor nucleus and analyzed the changes in proinflammatory cytokines and the blood-brain barrier. Peripheral nerve transection led to a rapid influx of CD3-, CD11a (alphaL, LFA1alpha)- and CD44-immunoreactive T-cells into the axotomized mouse facial motor nucleus, with a first, low-level plateau 2-4 d after injury, and a second, much stronger increase at 14 d. These T-cells frequently formed aggregates and exhibited typical cleaved lymphocyte nuclei at the EM level. Immunohistochemical colocalization with thrombospondin (TSP), a marker for phagocytotic microglia, revealed aggregation of the T-cells around microglia removing neuronal debris. The massive influx of lymphocytes at day 14 was also accompanied by the synthesis of mRNA encoding IL1beta, TNFalpha, and IFN-gamma. There was no infiltration by the neutrophil granulocytes, and the intravenous injection of horseradish peroxidase also showed an intact blood-brain barrier. However, mice with severe combined immunodeficiency (SCID), which lack differentiated T- and B-cells, still exhibited infiltration with CD11a-positive cells. These CD11a-positive cells also aggregated around phagocytotic microglial nodules. In summary, there is a site-selective infiltration of activated T-cells into the mouse CNS during the retrograde reaction to axotomy. The striking aggregation of these lymphocytes around neuronal debris and phagocytotic microglia suggests an important role for the immune surveillance during neuronal cell death in the injured nervous system.

Fig. 1.

CD3 immunohistochemistry in the normal and axotomized mouse facial motor nucleus. CD3-immunoreactive T-lymphocytes are absent in the normal facial nucleus (0d), but appear 1 d after axotomy (1d, arrows), reach a maximum at day 14, and disappear almost completely at 66 d (66d) after injury. The extent of the facial motor nucleus is indicated by thedotted lines in this and in the following figure (Fig.2). All magnifications 49×. Bottom right, Quantitative time course of CD3-positive cells in the axotomized and contralateral facial nuclei (mean ± SEM, n = 3 animals per time point). Note the early plateau of two to three labeled cells per section 1–4 d after axotomy, and a further 10-fold increase at day 14. No statistically significant increase on the contralateral side.

Fig. 2.

Distribution of CD3-immunoreactive T-lymphocytes in the axotomized facial motor nucleus 14 d after injury. A, Diffuse distribution. B, A rare perivascular infiltrate (thin arrow) surrounding a large vessel (v). C, D, Focal aggregates of CD3-immunoreactive T-lymphocytes (arrows). Magnification, 49×.

Fig. 3.

A–D, Different stages of microglial nodules in the mouse facial motor nucleus 14 d after injury in normal B6C3 mice; immunohistochemistry (brown staining) for TSP (A–C) and CD11a (D), 1 μm semithin araldite sections, methylene blue counterstain. A, Two activated microglia with slender TSP-immunoreactive processes (short arrows) adhere to an apoptotic neuron with nuclear chromatin condensation (long arrows). The arrowheads point to the TSP-negative astrocytes with clear and regular oval nuclei (also inB–D). B, Microglial phagocytosis of neuronal debris; strongly TSP-immunoreactive microglial nodule (short arrow) containing fragmented, methylene blue-counterstained cellular remnants (long arrows).C, Late stage TSP-immunoreactive microglial nodule (short arrow) consisting of three microglial cells after removal of the neuronal debris. The cellular structure of the TSP-immunoreactive nodules in this and the preceding micrograph (Fig.3B) is similar to that in E–H and Figure7C–F. D, Two microglial cells at the center of the nodule (m, long arrows) surrounded by CD11a-immunoreactive lymphocytes (short arrows).E–H, Colocalization of infiltrating lymphocytes and phagocytotic microglial nodules in the axotomized facial motor nucleus.E–G, Normal B6C3 mice, double immunofluorescence for thrombospondin and the T-lymphocyte markers CD3 (E), CD11a (F), and CD44 (G) 14 d after injury. Note the direct contact of T-lymphocytes (green) with the TSP-immunoreactive microglia (red). The CD44 immunoreactivity (G) is also present on the surface of axotomized motoneurons (Jones et al., 1997).H, SCID mouse facial motor nucleus, 14 d after injury. Apposition of CD11a-immunoreactive cells (green) on an IBA1-labeled microglial nodule (red). Magnification: A, 1140×;B–D, 900×; E–H, 950×.

Fig. 4.

Ultrastructural localization of CD11a- and CD3-immunoreactivity in the 14 d axotomized facial motor nucleus.A, CD11a immunostaining of a cellular aggregate, consisting of a degenerating neuron at the center, surrounded by microglia (M), astrocytes (A), and the CD11a-positive lymphocytes (L). These CD11a-positive cells frequently showed a clear cytoplasm, deeply cleaved nuclei, and ruffled, CD11-immunoreactive cell membranes (short arrows). The curved arrow points to phagosomes in a CD11a-negative cell process adhering to a CD11a-immunoreactive cell. These phagosomes are a common, characteristic feature in the phagocytotic microglial cells. Magnification, 5400×. B, C, CD3 immunoreactivity on the cell membrane of infiltrating T-lymphocytes (T). Note the typical cleaved or deformed lymphocyte nuclei. Adjacent vessels (V) and astrocytes (A) are unlabeled. Magnification:B, 5800×; C, 6800×.

Fig. 5.

Effects of timing and SCID-phenotype on lymphocyte infiltration. A, Effects of consecutive, bilateral axotomy. Bilateral infiltration of CD3 lymphocytes, 14 d after transection of the right and 3 d after transection of the left facial nerve. Note the ∼10-fold higher influx of lymphocytes on the 14 d axotomized side. *p < 0.001 in a paired, two-sided Student’s t test; mean ± SD (n = 4 animals). B, C, Infiltration of CD3- (B) and CD11a-immunoreactive cells (C) in normal and SCID mice in the BALB/c genetic background, 14 d after facial nerve transection (mean ± SEM,n = 5 animals). The SCID phenotype leads to a 98% decrease in the number of CD3-positive cells (p < 0.001) and a 60% decrease in the number of CD11a-positive cells (p < 0.01). Unpaired t test.

Fig. 6.

Immunohistochemical distribution of MHC class I (A, B), CD3 (C, D), and CD11a (E, F) immunoreactivity in normal (A, C, E) and SCID mice (B, D, F), 14 d after facial axotomy. A, B, Strong, focal increase of MHC class I immunoreactivity in the axotomized facial motor nuclei (right side). No specific immunoreactivity on the contralateral, unoperated side. Note the similar increase in MHC class I in normal and SCID animals. Magnification, 15×. C, D, CD3 immunoreactivity. Complete absence of specific staining in the SCID animal. E, F, CD11a immunoreactivity. Note the reduction in the number of CD11a-positive cells in the immunodeficient mouse. Magnification: C–F, 110×.

Fig. 7.

Facial motor nucleus, 14 d after axotomy, SCID mouse. A–F, Double immunofluorescence of microglial IBA1 immunoreactivity (red, A, C, E) with superimposed CD11a (B), CD11b (D), and MHC class I (F) labeling (green). Note the absence of colocalization of IBA1 with CD11a (B) and the colocalization with CD11b immunoreactivity (D, yellow). MHC class I immunoreactivity (F) is present both on IBA1-positive microglia (yellow) and on round, IBA1-negative cells (green, arrows). Thearrowheads point to the large microglial nodules. Magnification, 1050×.

Fig. 8.

Effects of axotomy on the blood–brain barrier (A–D) and the infiltration of neutrophil granulocytes, 14 d after facial nerve transection (E–G). A, Detection of HRP extravasation in area postrema and in the surrounding parenchyma.B, No gross HRP extravasation in the brain stem at the level of the facial motor nucleus. C, D, Higher magnification of the contralateral (C) and axotomized facial nucleus (D) only shows a specific HRP staining of the brain vasculature. E–H, Histochemical and immunohistochemical staining for neutrophil granulocytes in the spleen (E, G) and in the axotomized facial nucleus (F, H). E, F, Immunohistochemistry with a rat monoclonal antibody MCA771 against neutrophil granulocytes. G, H, Endogenous peroxidase. Both methods show the absence of granulocyte staining in the facial nucleus. Magnification: A, B, 13×;C–H, 53×.

Fig. 9.

RT-PCR detection of mRNA for IL1β, TNFα, and IFN-γ, 3 and 14 d after facial nerve transection in the axotomized facial motor nuclei (A1–A4) and on the contralateral side (C1–C4). Southern blotting with digoxigenin-end-labeled internal oligonucleotide probes. Day 3 shows a moderate increase for IL1β and TNFα, but not for IFN-γ mRNA. All animals showed a clear increase in IL1β, TNFα, and IFN-γ mRNA on the axotomized side at day 14. The constitutively expressed glucose 6-phosphate dehydrogenase (GAPDH) mRNA served as a recovery standard for RNA extraction, reverse transcription, and amplification with PCR.PC, Positive control with added synthetic cytokine RNA.NC, Negative control, omission of added RNA. The results for days 3 and 14 are from two separate experiments and preclude a direct comparison of the absolute amount of mRNA on the contralateral side at these two time points.