Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting

Abstract

Tumor necrosis factor (TNF)-dependent sites of action in the generation of autoimmune inflammation have been defined by targeted disruption of TNF in the C57BL/6 mouse strain. C57BL/6 mice are susceptible to an inflammatory, demyelinating form of experimental autoimmune encephalomyelitis (EAE) induced by the 35-55 peptide of myelin oligodendrocyte glycoprotein. Direct targeting of a strain in which EAE was inducible was necessary, as the location of the TNF gene renders segregation of the mutated allele from the original major histocompatibility complex by backcrossing virtually impossible. In this way a single gene effect was studied. We show here that TNF is obligatory for normal initiation of the neurological deficit, as demonstrated by a significant (6 d) delay in disease in its absence relative to wild-type (WT) mice. During this delay, comparable numbers of leukocytes were isolated from the perfused central nervous system (CNS) of WT and TNF-/- mice. However, in the TNF-/- mice, immunohistological analysis of CNS tissue indicated that leukocytes failed to form the typical mature perivascular cuffs observed in WT mice at this same time point. Severe EAE, including paralysis and widespread CNS perivascular inflammation, eventually developed without TNF. TNF-/- and WT mice recovered from the acute illness at the same time, such that the overall disease course in TNF-/- mice was only 60% of the course in control mice. Primary demyelination occurred in both WT and TNF-/- mice, although it was of variable magnitude. These results are consistent with the TNF dependence of processes controlling initial leukocyte movement within the CNS. Nevertheless, potent alternative mechanisms exist to mediate all other phases of EAE.

Figure 1

Natural history of EAE in WT and TNF^−/− C57BL/6 mice. (A) Mean clinical EAE scores (± SEM) of WT mice (•, n = 6) and TNF^−/− mice (○, n = 8) after immunization with MOG/CFA. The horizontal bar at days 13–15 indicates a time point of closer examination, referred to in Fig. 2 and in the text. (B) As a measure of the relative susceptibility of WT and TNF^−/− mice to neurological deficits induced by immunization with MOG, areas under the curves in A were determined. Results are representative of four separate time course studies.

Figure 2

Characterization of the early CNS inflammatory infiltrate in WT and TNF^−/− mice. (A) Leukocyte inflammation (CD45⁺) and VCAM-1 expression at day 15 (Fig. 1 A, horizontal bar). Tissue sections were derived from brain stem and cerebellum and are representative of tissues throughout the CNS of several WT and TNF ^−/− mice at this time point. (Inset) VCAM-1 expression of unimmunized C57BL/6J-strain CNS. Bar = 60 μm. (B) Total cell recoveries from the perfused CNS of normal and immunized mice over the course of disease. Replicates concentrated on the early disease phase when TNF^−/− mice were not showing signs of clinical disease. For simplicity, individual mice from days 13–15 are all shown at the day 15 time point. Each data point represents a single mouse. +, WT unimmunized. •, WT MOG immunized. ○, TNF^−/− MOG immunized. (C) T cell accumulation in the CNS of normal and MOG-immunized WT and TNF^−/− mice at day 15. Pooled cells obtained from two mice in each case were stained and analyzed by flow cytometry. Percentage of total cells in each preparation as defined by populations 1, 2, and 3 (see text) are shown in the inset. CD45⁻ TCR⁻ cells are undefined but would represent nonhematopoietically derived cells including neurons, endothelial cells, astrocytes, and oligodendrocytes.

Figure 2

Characterization of the early CNS inflammatory infiltrate in WT and TNF^−/− mice. (A) Leukocyte inflammation (CD45⁺) and VCAM-1 expression at day 15 (Fig. 1 A, horizontal bar). Tissue sections were derived from brain stem and cerebellum and are representative of tissues throughout the CNS of several WT and TNF ^−/− mice at this time point. (Inset) VCAM-1 expression of unimmunized C57BL/6J-strain CNS. Bar = 60 μm. (B) Total cell recoveries from the perfused CNS of normal and immunized mice over the course of disease. Replicates concentrated on the early disease phase when TNF^−/− mice were not showing signs of clinical disease. For simplicity, individual mice from days 13–15 are all shown at the day 15 time point. Each data point represents a single mouse. +, WT unimmunized. •, WT MOG immunized. ○, TNF^−/− MOG immunized. (C) T cell accumulation in the CNS of normal and MOG-immunized WT and TNF^−/− mice at day 15. Pooled cells obtained from two mice in each case were stained and analyzed by flow cytometry. Percentage of total cells in each preparation as defined by populations 1, 2, and 3 (see text) are shown in the inset. CD45⁻ TCR⁻ cells are undefined but would represent nonhematopoietically derived cells including neurons, endothelial cells, astrocytes, and oligodendrocytes.

Figure 3

Peak disease inflammation and demyelination. (A) Leukocyte infiltration and formation of perivascular cuffs. Sections were derived from spinal cord of animals harvested at day 19 (WT) and day 23 (TNF ^−/−) and stained for CD45. CD45⁺ filamentous processes within the CNS parenchyma (arrows) indicate activated microglia. Comparable infiltration was found at all levels of the spinal cord, brain stem, and cerebellum. Bar = 60 μm. (B) Primary demyelination in WT and TNF^−/− mice. Sections were derived from spinal cord of mice harvested at day 35 (WT) and day 40 (TNF ^−/−). Tissues from WT and TNF^−/− mice show a region of comparable perivenous (v) demyelination and gliosis beneath the meningeal surface (m). Arrows indicate naked (demyelinated) axons. In these mice, a similar histological picture was obtained at all levels of the spinal cord. Bar = 12 μm.