Distinct in vivo roles of colony-stimulating factor-1 isoforms in renal inflammation.

CSF-1, the major regulator of macrophage (Mphi) development, has three biologically active isoforms: a membrane-spanning, cell surface glycoprotein, a secreted glycoprotein, and a secreted proteoglycan. We hypothesized that there are shared and unique roles of individual CSF-1 isoforms during renal inflammation. To test this, we evaluated transgenic mice only expressing the cell surface or precursors of the secreted CSF-1 isoforms for Mphi accumulation, activation, and Mphi-mediated tubular epithelial cell (TEC) apoptosis during unilateral ureteral obstruction. The only difference between secreted proteoglycan and secreted glycoprotein CSF-1 isoforms is the presence (proteoglycan) or absence (glycoprotein) of an 18-kDa chondroitin sulfate glycosaminoglycan. We report that 1) cell surface CSF-1 isoform is sufficient to restore Mphi accumulation, activation, and TEC apoptosis to wild-type levels and is substantially more effective than the secreted CSF-1 isoforms; 2) the chondroitin sulfate glycosaminoglycan facilitates Mphi accumulation, activation, and TEC apoptosis; 3) increasing the level of secreted proteoglycan CSF-1 in serum amplifies renal inflammation; and 4) cell-cell contact is required for Mphi to up-regulate CSF-1-dependent expression of IFN-gamma. Taken together, we have identified central roles for the cell surface CSF-1 and the chondroitin sulfate chain on secreted proteoglycan CSF-1 during renal inflammation.

Self-peptides that bind with low affinity to the diabetes-associated I-A(g7) molecule readily induce T cell tolerance in non-obese diabetic mice.

Although non-obese diabetic (NOD) mice spontaneously develop T cell autoimmunity, it is not clear whether this phenomenon results from a defect in tolerance to self-Ag. Furthermore, as autoimmunity has been postulated to result from T cell responses directed toward self-peptides that bind with low affinity to NOD I-A(g7) MHC class II molecules, it is important to determine whether the expression of such peptides induces tolerance. We have constructed NOD transgenic (Tg) mice expressing the Leishmania antigen receptor for C kinase (LACK) Ag in either the thymus or pancreatic beta cells. We identified LACK peptides that were the targets of T cells in LACK-immunized NOD mice while binding to I-A(g7) with low affinity. While CD4(+) T cells from NOD mice secreted IFN-gamma, IL-4, IL-5 and IL-10 in response to LACK, those from LACK-expressing Tg mice secreted reduced levels of cytokines. Experiments using peptide/MHC multimers showed that LACK-expressing Tg mice exhibited self-reactive CD4(+) T cells with impaired proliferation capabilities. Hence, even self-peptides that bind to I-A(g7) with low affinity can induce tolerance in NOD mice. This result is important in light of the commonly held hypothesis that T cells reacting to peptides that bind to MHC with low affinity escape tolerance induction and cause autoimmunity.

Ex vivo analysis of thymic CD4 T cells in nonobese diabetic mice with tetramers generated from I-A(g7)/class II-associated invariant chain peptide precursors.

The MHC determines susceptibility and resistance to type 1 diabetes in humans and nonobese diabetic (NOD) mice. To investigate how a disease-associated MHC molecule shapes the T cell repertoire in NOD mice, we generated a series of tetramers from I-A(g7)/class II-associated invariant chain peptide precursors by peptide exchange. No CD4 T cell populations could be identified for two glutamic acid decarboxylase 65 peptides, but tetramers with a peptide mimetic recognized by the BDC-2.5 and other islet-specific T cell clones labeled a distinct population in the thymus of young NOD mice. Tetramer-positive cells were identified in the immature CD4(+)CD8(low) population that arises during positive selection, and in larger numbers in the more mature CD4(+)CD8(-) population. Tetramer labeling was specific based on the use of multiple control tetramers, including one with a single amino acid analog peptide in which a critical TCR contact residue was substituted. The T cell population was already present in the thymus of 2-wk-old NOD mice before the typical onset of insulitis and was detected in B10 mice congenic for the NOD MHC locus, but not B10 control mice. These results demonstrate that a T cell population can expand in the thymus of NOD mice to levels that are at least two to three orders of magnitude higher than estimated for a given specificity in the naive T cell pool. Based on these data, we propose a model in which I-A(g7) confers susceptibility to type 1 diabetes by biasing positive selection in the thymus and later presenting peptides from islet autoantigens to such T cells in the periphery.

Detection of autoreactive myelin proteolipid protein 139-151-specific T cells by using MHC II (IAs) tetramers.

Detection of autoreactive T cells using MHC II tetramers is difficult because of the low affinity of their TCR. We have generated a class II tetramer using the IA(s) class II molecule combined with an autoantigenic peptide from myelin proteolipid protein (PLP; PLP(139-151)) and used it to analyze myelin PLP(139-151)-reactive T cells. Using monomers and multimerized complexes labeled with PE, we confirmed the specificity of the reagent by bioassay and flow cytometry. The IA(s) tetramers stimulated and stained the PLP(139-151)-specific 5B6 TCR transgenic T cells and a polyclonal cell line specific for PLP(139-151), but not a control T cell line specific for PLP(178-191). We used this reagent to optimize conditions to detect low affinity autoreactive T cells. We found that high pH ( approximately 8.0) and neuraminidase treatment enhances the staining capacity of PLP(139-151) tetramer without compromising specificity. Furthermore, we found that induction of calcium fluxing by tetramers in T cells may be used as a sensitive measure to detect autoreactive T cells with a low affinity. Taken together, the data show that the tetrameric reagent binds and stimulates PLP(139-151)-reactive T cells with specificity. This tetrameric reagent will be useful in studying the evolution of PLP(139-151)-specific repertoire in naive mice and its expansion during the autoimmune disease experimental autoimmune encephalomyelitis.