Roel C. van der Veen

EXTENSIVE PEROXYNITRITE ACTIVITY DURING PROGRESSIVE STAGES OF CENTRAL NERVOUS SYSTEM INFLAMMATION

Nitric oxide (NO) production has been associated with disease activity in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). This free radical can be transformed by superoxide to peroxynitrite, an extremely toxic oxidant which causes lipid peroxidation. In addition, peroxynitrite nitrates tyrosine residues, resulting in nitrotyrosine, which can be identified immunohistochemically. The results of this study indicate that peroxynitrite is formed very early during EAE development, correlating with clinical disease activity. Nitrotyrosine-positive cells display a widespread distribution in brain and spinal cord during severe disease and are associated with both perivascular infiltrates and parenchymal sites. Double-staining procedures demonstrated that a subpopulation of CD11b-positive cells (macrophages/microglia) reacted with nitrotyrosine antibodies. Immunostaining for inducible NO synthase demonstrated a similar distribution as nitrotyrosine staining. These experiments indicate that peroxynitrite is formed during progressive stages of disease activity.

Encephalitogenic Th1 cells are inhibited by Th2 cells with related peptide specificity: Relative roles of interleukin (IL)-4 and IL-10

Cytokines secreted by T-helper type 2 (Th2) cells inhibit the antigen-induced stimulation of type 1 (Th1) helper T cells. To study this form of regulation in an autoimmune disease model, the cytokines secreted by a Th2 clone specific for the encephalitogenic proteolipid protein (PLP) peptide 139-151 were tested for their ability to inhibit proliferation of an encephalitogenic Th1 clone specific for an epitope contained within the same peptide. Cytokines, produced by stimulation of the Th2 clone with CD3-specific monoclonal antibodies (mAbs), inhibited proliferation of the Th1 clone when stimulated by antigen and splenic antigen-presenting cells (APC). Inhibition was, however, not antigen-specific since cytokines released upon stimulation of an unrelated Th2 clone were also inhibitory. Inhibition was found to be caused by effects on either antigen presentation or co-stimulatory activity of the APC and not by direct effects on the Th1 cells. MAbs for the two major regulatory Th2 cytokines were used to identify the inhibitory component secreted by activated Th2 cells. Interleukin-10 (IL-10)-specific mAb abolished the inhibitory effect, while mAb specific for IL-4 had no effect on inhibition. The addition of recombinant IL-4 (rIL-4) and rIL-10 confirmed that inhibition of Th1 proliferation was due to secretion of IL-10 by the Th2 clone and its subsequent effects on APC. The studies described here demonstrate that PLP-specific Th2 cells which recognize peptide 139-151 inhibit encephalitogenic Th1 cells which respond to an epitope on the same peptide. This phenomenon may be important for local, antigen-specific regulation of inflammation in the central nervous system.

Superoxide Prevents Nitric Oxide-Mediated Suppression of Helper T Lymphocytes: Decreased Autoimmune Encephalomyelitis in Nicotinamide Adenine Dinucleotide Phosphate Oxidase Knockout Mice

NO, which suppresses T cell proliferation, may be inactivated by superoxide (O2−) due to their strong mutual affinity. To examine this possibility, preactivated Th clones were cocultured with stimulated macrophages. PMA neutralized the inhibitory activity of NO, which was dependent on extracellular O2− production. In contrast, macrophages from p47phox −/− (pKO) mice, which lack functional NADPH oxidase, retained their NO-dependent inhibition of T cell proliferation upon stimulation with PMA, indicating that NADPH oxidase is the major source of NO-inactivating O2− in this system. To examine the NO-O2− interaction in vivo, the role of NADPH oxidase in experimental autoimmune encephalomyelitis was studied in pKO mice. No clinical or histological signs were observed in the pKO mice. Neither a bias in Th subsets nor a reduced intensity of T cell responses could account for the disease resistance. Although spleen cells from pKO mice proliferated poorly in response to the immunogen, inhibition of NO synthase uncovered a normal proliferative response. These results indicate that NO activity may play a critical role in T cell responses in pKO mice and that in normal spleens inhibition of T cell proliferation by NO may be prevented by simultaneous NADPH oxidase activity.

Nitric oxide and T helper cell immunity

In this article, the controversial role of nitric oxide (NO) in T helper (Th) cell activation and T-cell-dependent immunity will be discussed with an emphasis on immunosuppression by NO. NO is generated by antigen-presenting cells (APC) during the process of antigen presentation to T cells. In mouse models, activation of the inducible NO synthase (iNOS) in APC is triggered by Th1-cell-derived IFN-γ, in combination with other soluble or membrane-associated T-cell factors. The NO so-produced inhibits T-cell proliferation, while it does not inhibit T cell cytokine production. NO blocks T-cell proliferation during G1/S transition. In mouse models of T-cell-mediated autoimmunity such as myelin antigen-induced EAE, the disease is exacerbated by genetic deletion of iNOS, indicating that NO suppresses T-cell-mediated immunity in vivo. Recent studies reveal that interaction with superoxide diminishes the T-cell regulatory activity of NO. The role for NADPH oxidase as a source for NO-inhibiting superoxide is discussed. In conclusion, NO plays an important regulatory role in the induction phase of T-cell-mediated immunity. Superoxide may enhance T-cell-mediated immunity by preventing the immunosuppressive activity of NO.

Mycobacteria-induced Gr-1+ subsets from distinct myeloid lineages have opposite effects on T cell expansion

Similar to the regulation of vasodilation, the balance between NO and superoxide (O2–) regulates expansion of activated T cells in mice. Reduction of suppressive NO levels by O2– is essential for T cell expansion and development of autoimmunity. In mice primed with heat‐killed Mycobacterium, a splenocyte population positive for Gr‐1 (Ly‐6G/C) is the exclusive source of both immunoregulatory free radicals. Distinct Gr‐1+ cell subpopulations were separated according to Ly‐6G expression. In culture with activated T cells, predominantly monocytic Ly‐6G− Gr‐1+ cells produced T cell‐inhibitory NO but no O2–. However, mostly granulocytic Ly‐6G+ cells produced O2– simultaneously but had no measurable effect on proliferation. Recombination of the two purified Gr‐1+ subpopulations restored controlled regulation of T cell proliferation through NO and O2– interaction. Coculture of p47phox−/− and inducible NO synthase−/− Gr‐1+ cells confirmed this intercellular interaction. These data suggest that bacterial products induce development of distinct Gr‐1+ myeloid lineages, which upon stimulation by activated T cells, interact via their respective free radical products to modulate T cell expansion.

Contrasting roles for nitric oxide and peroxynitrite in the peroxidation of myelin lipids

Peroxynitrite is formed by the reaction of nitric oxide (NO) and superoxide. Since widespread peroxynitrite activity was observed during experimental allergic encephalomyelitis (EAE), the effect of this strong lipid-peroxidizing agent on myelin integrity was examined. Incubation of myelin suspensions with the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) resulted in the formation of the lipid peroxidation product, malondialdehyde (MDA). MDA formation was inhibited in the presence of butylated hydroxytoluene, which interrupts the progression of the lipid peroxidation chain reaction. Superoxide dismutase inhibited the effect of SIN-1, which indicates a role for superoxide, and contradicts a role for its dismutation product, hydrogen peroxide. The latter was confirmed by the failure of the catalase to inhibit MDA formation. Neither NO nor superoxide alone induced significant MDA formation in myelin, indicating that peroxynitrite formation is required for myelin-lipid peroxidation. Interestingly, NO actually inhibited lipid peroxidation in myelin, as demonstrated using simple NO donors. On the other hand, the simultaneous production of superoxide, as achieved with the NO-donor SIN-1, negated the inhibitory effect of NO. Finally, the production of isoprostanes, novel products generated during lipid peroxidation, was examined. Peroxynitrite-induced peroxidation of myelin resulted in isoprostane formation. Furthermore, increased levels of F2-isoprostanes and neuroprostanes were observed in spinal cords of mice during early progressive stages of autoimmune encephalomyelitis.

Fine-specificity differences in the recognition of an encephalitogenic peptide by T helper 1 and 2 cells

The lymphokine production of two T-cell clones, which both recognize epitopes within the encephalitogenic 139-151 sequence of myelin proteolipid protein, was examined after stimulation with immobilized antibodies to the CD3 moiety of the T-cell-receptor complex. Clone A1 produced interleukin (IL)-2 and interferon (IFN)-gamma, but no IL-4, while clone D5 produced IL-4, but no IL-2 or IFN-gamma. A1 therefore belongs to the T-helper type 1 (Th1) subset, while D5 is a Th2 clone. In addition, the Th1 clone induced severe experimental allergic encephalomyelitis (EAE), while the Th2 clone did not induce any signs of EAE. Synthetic peptides were used to demonstrate that these clones recognized slightly different epitopes within the 139-151 sequence. Histidine 139 was shown to be optimal for the stimulation of the Th2 clone, while the presence of this residue inhibited the stimulation of the Th1 clone. Th2 cells specific for an encephalitogenic peptide may be important in the regulation of encephalitogenic Th1 cells.

Chronic experimental allergic encephalomyelitis and antibody responses in rabbits immunized with bovine proteolipid apoprotein.

A chronic form of experimental allergic encephalomyelitis can be produced by sensitization of rabbits with bovine myelin proteolipid apoprotein (PLP). To investigate the humoral immune response in this model, serum PLP antibodies were determined by enzyme-linked immunosorbent and dot immunobinding assays. In an initial experiment, 3 PLP-sensitized rabbits with severe chronic experimental allergic encephalomyelitis had a positive antibody response whereas 3 with mild disease, or with no visible clinical disease, had no detectable antibodies against PLP. In a second experiment, 3 rabbits were preimmunized with PLP in incomplete Freunds adjuvant, followed by a single immunization with PLP in complete Freunds adjuvant. These animals developed chronic experimental allergic encephalomyelitis with different progression rates, although all eventually became severely paralyzed. In both experiments the anti-PLP response was maximal before or immediately after disease onset and tended to decline during disease progression. The degree of the anti-PLP response correlated with clinical and histologic disease severity. These data suggest a possible role for humoral factors in the modulation of the chronic EAE induced in PLP-immunized rabbits.

Immune processing of proteolipid protein by spleen-cell subsets

Reel C. van d e r Veen, John L. Trot te r , Jud i th A. Kapp , Washin~un University School of Medicine, St. Louis, M e 63110, USA Myelin proteolipid protein (PLP) has one recognized epitope wi*.hin the 139 -151 amino acid sequence (PLP-EP), that is encephalitogenic for SJL mice. In the current study, the proees~ing of P IP by different antigen-presenting cells (APC) was examined. In order to study whether PIP requires processing before its presentation by APC, PiP-pulsed and fixed APC were shown to stinmlate PiP-specific T cells. However, the addition of P IP to unpl~.lsed, fixed APC resulted in the absence ofT-cell stimulation, while the viability of these fixed PEC to bind antigenic peptide and efficiently present it to T cells was demonstrated by their ability to use a synthetic peptide for the stimulation of T cells. In order to study possible processing differences among APC subsets, spleen cells were fractionated by adherence to plastic, and their respective APC a:tivities were studied separately. Although both adherent and non-adherent spleen cells stimulated PLP-specific T-cell lines, the non-adherent APC were unable to stimulate an encephalitogenic T-cell clone with PIP. The specificity of the limited APC ability by non-adherent spleen cells was indicated by similar results us;.ng a second T-cell clone with specificity for the encephalitogenic pepfide. In contrast to these PLP-EP specific clones, a T-cell clone specific for a separate, unidentified epitope on PIP was stimulated by non-adherent APC efficiently. In contrast, stimulation of PLP-EP specific T-cell clones by nowadherent APC did occur when the synthetic peptide instead of intact P IP was used as antigen, suggesting a defect in PIP processing by the non-adherent APC. Further fractionation of the irradiated, non-adherent spleen cells demonstrated that their activity was limited to a nonB, and non-T cell subfraction. These results indicate that a subpopulation of spleen APC is unable to process PIP efficiently, and more specifically, may be unable to process a fragment containing the 139! 51 sequence in PIP, while other fragments of P IP can be processed and suby, e~luently presented efficiently. 159