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

Körner H, Riminton DS, Strickland DH, Lemckert FA, Pollard JD, Sedgwick JD - J. Exp. Med. (1997)

Bottom Line: In this way a single gene effect was studied.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.

View Article: PubMed Central - PubMed

Affiliation: Centenary Institute of Cancer Medicine and Cell Biology, Royal Prince Alfred Hospital, Sydney, New South Wales, 2050 Australia.

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.

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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.
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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.

Mentions: Since TNF appeared to be an essential participant in the early events leading to the development of EAE, a series of experiments was performed to establish how the absence of TNF affected the CNS inflammatory process, using the time point at which the discrepancy between the clinical scores of TNF−/− and WT mice was maximal (Fig. 1 A, days 13–15, horizontal bar). Immunohistological analysis of mice at this time revealed small but frequently detectable accumulations of leukocytes (CD45+) in TNF−/− mice (Fig. 2 A, upper panels), but a marked reduction in discrete perivascular cuffs of leukocytes relative to WT. On the other hand, no detectable accumulations of leukocytes (CD45+) were found in the CNS of normal nonimmunized mice (data not shown). Clear evidence for the existence of immunological activation within the CNS was revealed by staining for the adhesion molecule VCAM-1 (Fig. 2 A, lower panels), which was substantially upregulated on vascular endothelium throughout the CNS of TNF−/− mice.


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

Körner H, Riminton DS, Strickland DH, Lemckert FA, Pollard JD, Sedgwick JD - J. Exp. Med. (1997)

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.
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Related In: Results  -  Collection

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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.
Mentions: Since TNF appeared to be an essential participant in the early events leading to the development of EAE, a series of experiments was performed to establish how the absence of TNF affected the CNS inflammatory process, using the time point at which the discrepancy between the clinical scores of TNF−/− and WT mice was maximal (Fig. 1 A, days 13–15, horizontal bar). Immunohistological analysis of mice at this time revealed small but frequently detectable accumulations of leukocytes (CD45+) in TNF−/− mice (Fig. 2 A, upper panels), but a marked reduction in discrete perivascular cuffs of leukocytes relative to WT. On the other hand, no detectable accumulations of leukocytes (CD45+) were found in the CNS of normal nonimmunized mice (data not shown). Clear evidence for the existence of immunological activation within the CNS was revealed by staining for the adhesion molecule VCAM-1 (Fig. 2 A, lower panels), which was substantially upregulated on vascular endothelium throughout the CNS of TNF−/− mice.

Bottom Line: In this way a single gene effect was studied.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.

View Article: PubMed Central - PubMed

Affiliation: Centenary Institute of Cancer Medicine and Cell Biology, Royal Prince Alfred Hospital, Sydney, New South Wales, 2050 Australia.

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.

Show MeSH
Related in: MedlinePlus