Experimental autoimmune encephalomyelitis in the rat.

There are several diverse rat models of experimental autoimmune encephalomyelitis (EAE) that can be used to investigate the pathogenesis and regulation of autoimmunity against CNS myelin. The disease course of these models ranges from an acute monophasic disease with limited demyelination to a chronic relapsing or chronic progressive course marked by severe demyelination. These models enable the study of encephalitogenic T cells and demyelinating antibody specific for major neuroantigens such as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein (PLP), among other important CNS autoantigens. Overall, this unit provides an overview of common methods for induction of active and passive EAE, assessment and analysis of clinical disease, preparation and purification of myelin basic protein, and derivation of neuroantigen-specific rat T cell lines. This unit also provides a brief discussion of the basic characteristics of these models.

Myelin basic protein-reactive T cells persist in an inactive state in the bone marrow of Lewis rats that have recovered from autoimmune encephalomyelitis.

Lewis rats immunized with guinea pig myelin basic protein residues 68-86 develop acute experimental autoimmune encephalomyelitis and recover. The predominant T cell receptor expressed by the encephalitogenic T cells is TCRBV8S2. They persist in bone marrow many weeks after recovery. CD3 is down-regulated, but >90% express CD4. They fail to proliferate to GPMBP68-86 unless a nitric oxide synthase inhibitor is added to the cultures. Perhaps these are memory T cells that are maintained in a suppressed state in BM by a nitric oxide-dependent mechanism.

Characterization of a subset of bone marrow-derived natural killer cells that regulates T cell activation in rats.

We report that bone marrow-derived natural killer (BMNK) cells from DA or F344 rats inhibit PMA/ionomycin-induced T cell proliferation. These NK-regulatory cells are NKR-P1A(dim), whereas a minor subpopulation is NKR-P1A(bright). Only the NKR-P1A(dim) BMNK cells inhibit T cell proliferation. If activated with rat Con A supernatant, the NKR-P1A(dim) cells become NKR-P1A(bright) and lose the ability to inhibit T cell proliferation. In contrast to BMNK cells, all DA and F344 rat NK cells isolated from the blood, spleen, cervical, or mesenteric lymph nodes or Peyer's patches are NKR-P1A(bright) and lack the ability to inhibit T cell proliferation. Inhibition of T cell proliferation correlates with significant down-regulation of CD3, suggesting that this may be the mechanism through which the NKR-P1A(dim) cells mediate suppression. The nitric oxide synthase inhibitor N(G)-monomethyl-arginine acetate-abrogated NKR-P1A(dim) cell inhibition of T cell proliferation. We conclude that rat bone marrow NKR-P1A(dim) cells represent a unique population that may play a role in maintaining immune homeostasis by regulating the clonal expansion of activated T cells.

Synergistic interaction between Toll-like receptor agonists is required for induction of experimental autoimmune encephalomyelitis in Lewis rats.

To investigate whether TLR agonists can replace mycobacteria in adjuvant to induce EAE in Lewis rats, we immunized rats with MBP peptide (MBP(68-86)) in IFA, supplemented with TLR agonists. Rats immunized with MBP(68-86) plus CpG-ODN or LPS in IFA did not develop EAE. In contrast, rats immunized with MBP(68-86) plus CpG-ODN and LPS in IFA developed clinical EAE. Spleen cells proliferated and secreted IFN-gamma in response to MBP(68-86), and secreted IL-6 and IL-12p40 in response to CpG-ODN and LPS. However, rats immunized with MBP(68-86) plus CpG-ODN and PolyI:C, a TLR3 agonist, did not develop EAE. We conclude that selected combinations of TLR agonists can facilitate the induction of EAE by MBP peptide via the innate immune system.

Molecular mimicry and horror autotoxicus: do chlamydial infections elicit autoimmunity?

All species of the order Chlamydiales are obligate intracellular eubacterial pathogens of their various hosts. Two chlamydial species, Chlamydia trachomatis and Chlamydia pneumoniae, are primarily human pathogens, and each is known to cause important diseases. Some strains of C. trachomatis are sexually transmitted and frequently cause severe reproductive problems, primarily in women. Other strains of the organism serve as the aetiological agents for blinding trachoma, still the leading cause of preventable blindness in underdeveloped nations. C. pneumoniae is a respiratory pathogen known to cause community-acquired pneumonia. Importantly, both organisms engender an immunopathogenic response in the human host, and both have been associated with widely diverse, relatively common and currently idiopathic chronic diseases, most of which include an important autoimmune component. In this article, we explore the available experimental data regarding the possible elicitation of autoimmunity in various contexts by chlamydial infection, and we suggest several avenues for research to explore this potentially important issue further.

NK cells inhibit T cell proliferation via p21-mediated cell cycle arrest.

NK cells have been shown to influence immune responses via direct interaction with cells of the adaptive immune system, such as dendritic cells, B cells, and T cells. A role for NK cells in down-regulation of T cell responses has been implicated in several studies; however, the underlying mechanism of this suppression has remained elusive. In this study we show that dark Agouti rat NK cells inhibit syngeneic T cell proliferation via up-regulation of the cell cycle inhibitor, p21, resulting in a G0/G1 stage cell cycle arrest. The inhibition is cell-cell contact dependent, reversible, and Ag nonspecific. Interestingly, NK cells do not inhibit IL-2 secretion or IL-2R up-regulation and do not induce T cell death. Thus, our results show that NK cells do not affect early T cell activation events, but specifically inhibit T cell proliferation by direct interaction with T cells. Our findings suggest that NK cells may play an important role in maintaining immune homeostasis by directly regulating clonal expansion of activated T cells. This novel mechanism of T cell regulation by NK cells provides insight into NK cell-mediated regulation of adaptive immunity and provides a mechanistic link between NK cell function and suppression of T cell responses.

Substance P regulates natural killer cell interferon-gamma production and resistance to Pseudomonas aeruginosa infection.

Studies have shown that after Pseudomonas aeruginosa (P. aeruginosa) corneal infection, BALB/c mice that are capable of resolving the disease, locally produce IFN-gamma. As T cells are not detected in the infected cornea of these mice, antibody depletion was used to test whether NK cells produce the cytokine. After depletion, decreased corneal IFN-gamma mRNA and increased disease severity, bacterial load, and PMN infiltrate resulted. Further work determined if substance P (SP), a pro-inflammatory neuropeptide, participated in regulation of this response. To this end, mice were treated with the SP antagonist, spantide I that blocks SP interaction with neurokinin-1, its major receptor. The treatment significantly decreased corneal IFN-gamma and IL-18 protein levels and corneal perforation resulted. In vitro experiments using isolated splenic NK cells confirmed their ability to respond to IL-18 and SP and to secrete IFN-gamma protein. We conclude: that for development of the BALB/c resistance response, NK cells are required to produce IFN-gamma; that the cells express the neurokinin-1 receptor; and that SP directly regulates IFN-gamma production through this receptor. The data suggest a unique link between the nervous system and development of innate immunity in the cornea.

Autoreactive T cells persist in rats protected against experimental autoimmune encephalomyelitis and can be activated through stimulation of innate immunity.

Lewis rats can be rendered unresponsive to experimental autoimmune encephalomyelitis by immunization with myelin basic protein (MBP), or MBP68-86, the dominant encephalitogenic MBP epitope for this strain, administered in IFA. However, protected rats harbor potentially encephalitogenic T cells, which are maintained in an inactive state. We investigated whether these quiescent effector cells could be activated in vitro. Although these T cells respond poorly to MBP68-86, they proliferate vigorously whether cocultured with MBP68-86 and either IL-2 or IL-12, suggesting that the T cells are in a state of anergy. Moreover, we could activate these anergic T cells with peptide and cytosine-guanine dinucleotide (CpG) oligonucleotide, but not control oligonucleotide, suggesting that products of the innate immune response are capable of activating anergic autoreactive T cells. The activated T cells produced the proinflammatory cytokine, IFN-gamma in response to IL-12, and IL-6 was secreted in response to CpG oligonucleotide. IL-6 has been reported to play a role in T cell activation by blocking T regulatory/suppressor (Treg) cell-mediated suppression through a Toll-like receptor-dependent pathway. However, anti-IL-6 mAb did not block CpG activation of the anergized cells. In contrast, anti-TGF-beta(1) Ab released the unresponsive T cells from the anergic state in the presence of MBP68-86, whereas TGF-beta(1) inhibited proliferation of MBP68-86- plus CpG-activated T cells. Because TGF-beta(1) has previously been implicated in Treg activity, this finding is consistent with a role for Treg cells in maintaining autoreactive T cells in the anergic state.

MHC class II peptide flanking residues of exogenous antigens influence recognition by autoreactive T cells.

Molecular mimicry between exogenous microbial antigens and self-epitopes has been proposed as a triggering mechanism for autoimmune diseases for many years. We reported that a peptide from a protein specific to Chlamydia pneumoniae (Cpn0483) which shares a motif with the dominant encephalitogenic epitope of the self-antigen, rat myelin basic protein (rat68-86), elicits experimental autoimmune encephalomyelitis (EAE) in Lewis rats. We recently observed that rat68-86 utilizes aspartic acid (D) and arginine (R) in the common motif as primary and secondary TCR contacts, respectively. In contrast, the encephalitogenic activity of Cpn0483 is dependent on R and the C-terminal asparagine (N), which flanks the MHC class II-P9 anchor residue. Thus, rat68-86 and Cpn0483 share a common motif, are encephalitogenic and are both restricted by MHC class II RT1.B(l). T cells from rats immunized with the encephalitogenic Cpn0483 peptide proliferated to the priming peptide as well as to the non-encephalitogenic CpnN>A analog. However, CpnN>A-primed T cells did not respond to the native Cpn0483 peptide. We conclude that the MHC-flanking C-terminal asparagine residue markedly influences T cell recognition by the chlamydial peptide.

Infectious agents and multiple sclerosis--are Chlamydia pneumoniae and human herpes virus 6 involved?

A good deal of evidence suggests an infectious component in the development of multiple sclerosis (MS) and, to date, some 20 bacteria and viruses have been associated with the disease. Recent independent sets of studies have implicated the respiratory bacterium Chlamydia pneumoniae and human herpes virus 6 (HHV-6) in the pathogenesis of MS. However, as is the case for essentially all earlier microbial associations, experimental evidence linking either this bacterium or this virus to MS is equivocal. We review the published reports concerning involvement of C. pneumoniae and HHV-6 in MS, and data relating to possession of the APOE epsilon 4 allele, which some studies indicate might influence how these or other pathogens affect disease genesis. Based on the large set of inconsistent observations available and given important new information regarding the neuropathology of MS, we contend that no conclusion is possible at this point regarding the potential role of either C. pneumoniae or HHV-6 in MS. We therefore propose future studies that should clarify whether, and if so how, these and other organisms function in the pathogenesis of this disease.

Human herpesvirus 6 and Chlamydia pneumoniae as etiologic agents in multiple sclerosis - a critical review.

Multiple sclerosis (MS) is thought by many investigators to have an infectious component, and several microorganisms have been associated with the disease during the last three decades. Recent studies have implicated both human herpesvirus 6 (HHV-6) and the obligate intracellular bacterium Chlamydia pneumoniae in the etiology of MS. As with earlier studies of other potential agents, however, evidence linking either of these organisms to the disease is equivocal. In this article, we review data for and against involvement of HHV-6 and C. pneumoniae in MS, as well as evidence concerning auxiliary factors, such as possession of the APOE epsilon4 allele, which may influence the role of these organisms in pathogenesis. Further, we suggest several lines of investigation that should clarify whether either or both pathogens are associated meaningfully with this disease.

Experimental autoimmune encephalomyelitis in the rat.

This unit details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Detailed materials and methods required for the purification of both PLP and MBP are also described. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains.