Nitric oxide contributes to resistance of the Brown Norway rat to experimental autoimmune encephalomyelitis

Abstract

The Brown Norway (BN) rat is reported to be resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and a number of mechanisms have been suggested to explain this resistance. In work reported here we provide evidence that such resistance in the BN rat can be accounted for, at least in part, by their ability to produce higher levels of nitric oxide (NO) than susceptible strains of rats. Spleen cells from the BN rat make significantly more NO following in vitro stimulation than do cells from the Lewis or PVG rat and following in vivo immunization using complete Freund's adjuvant (CFA) the BN rat makes substantially more NO than either susceptible strain. If carbonyl iron is used as adjuvant in vivo there is no increase in NO levels in the BN rat and they are rendered highly susceptible to EAE. Immunizing with CFA simultaneously with neuroantigen and carbonyl iron drives up NO levels and the resistance is restored. EAE produced using carbonyl iron is characterized by extensive macrophage/microglia presence in the central nervous system lesions of the BN rat yet the cytokine profile in the lymph nodes does not differ from that in the EAE Lewis rats.

Figure 1

RNI levels in the serum (A) of PVG (hashed bars) and BN (black bars) rats following immunization with MBP-CFA are significantly increased when compared to the levels in Lewis rats (white bars) (significance of 0.006, analysis of variance Post-Hoc Test) but the BN levels do not differ from the PVG (except at day 1 where they are in fact lower). RNI levels in the urine (B) of the BN are significantly greater than either the Lewis or PVG (significance of 0.006, analysis of variance Post-Hoc Test) suggesting that the BN rat does make higher levels than the PVG but the RNI are excreted (5 animals/group/time period).

Figure 2

No clinical disease (black circle) and no change in body weight (gray circle) is seen in the BN rats immunized with SCH-CFA (A) but there is an increase in the serum RNI levels (histogram) (7 rats/group). Following immunization with SCH-carbonyl iron, however, (B) there is no increase in the serum RNI levels and the animals develop severe EAE accompanied by significant weight loss (11 rats/group).

Figure 3

Immunohistochemistry demonstrating iNOS expression in draining lymph nodes (A and D), spleen (B and E), and peritoneal exudate cells (C and F) of BN rats immunized with either SCH-CFA (top row) or SCH-CI (bottom row). The iNOS positivity in the tissues is brown-orange while in the cytospin of peritoneal cells it is brown-pink. Arrows in F point to carbonyl iron particles. Magnification, ×25.

Figure 4

Histopathology (H&E counterstain) of lumbar spinal cord from Lewis (A-D) and BN (E-H) rats immunized with either SCH-CFA (A and E) or SCH-CI (B to D and F to H). Lewis rats killed at day 12 after immunization with SCH-CFA have severe lesions in many parts of the spinal cord. Only the meninges are shown here (A). For comparison, BN rats immunized also with SCH-CFA have no disease and show no lesions in the meninges (E) or elsewhere. Immunization of both strains with SCH-CI led to severe disease and this was reflected in intense inflammatory lesion in meninges (B and F), white matter (C and G), as well as some lesions in the gray matter (D and H). These tissues were also stained for iNOS and arrows indicate positive-staining areas. Magnification, ×25.

Figure 5

Sections serial to those in Figure 4 stained immunohistochemically for ED-1⁺ cells (macrophages/microglia). Iit should be noted that although the quantitative comparison of lesions between the Lewis and BN when immunized with SCH-CI were very similar (Figure 4), there is a dramatic qualitative difference in lesions between the two rat strains with ED-1⁺ cells making up the majority of the inflammatory cell population in the BN (F to H) but not in the Lewis (B to D). Also note little iNOS expression (Figure 4) on this extensive macrophage/microglia infiltration. Magnification, ×25.