Richard Y. Yeh

We have identified and cloned a novel human cytokine with homology to cytokines of the interleukin-17 (IL-17) family, which we have termed human IL-17E (hIL-17E). With the identification of several IL-17 family members, it is critical to understand the in vivo function of these molecules. We have generated transgenic mice overexpressing hIL-17E using an apolipoprotein E (ApoE) hepatic promoter. These mice displayed changes in the peripheral blood, particularly, a 3-fold increase in total leukocytes consisting of increases in eosinophils, lymphocytes, and neutrophils. Splenomegaly and lymphoadenopathy were predominant and included marked eosinophil infiltrates and lymphoid hyperplasia. CCR3(+) eosinophils increased in the blood and lymph nodes of the transgenic mice by 50- and 300-fold, respectively. Eosinophils also increased 8- to 18-fold in the bone marrow and spleen, respectively. In the bone marrow, most of the eosinophils had an immature appearance. CD19(+) B cells increased 2- to 5-fold in the peripheral blood, 2-fold in the spleen, and 10-fold in the lymph nodes of transgenic mice, whereas CD4(+) T lymphocytes increased 2-fold in both blood and spleen. High serum levels of the cytokines IL-2, IL-4, IL-5, granulocyte colony-stimulating factor, eotaxin, and interferon gamma were observed. Consistent with B-lymphocyte increases, serum immunoglobulin (Ig) M, IgG, and IgE were significantly elevated. Antigenic challenge of the transgenic mice with keyhole limpet hemocyanin (KLH) resulted in a decrease in anti-KLH IgG accompanied by increases of anti-KLH IgA and IgE. In situ hybridization of transgenic tissues revealed that IL-17Rh1 (IL-17BR/Evi27), a receptor that binds IL-17E, is up-regulated. Taken together, these data indicate that IL-17E regulates hematopoietic and immune functions, stimulating the development of eosinophils and B lymphocytes. The fact that hIL-17E overexpression results in high levels of circulating eosinophils, IL-4, IL-5, eotaxin, and IgE suggests that IL-17E may be a proinflammatory cytokine favoring Th2-type immune responses.

Lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-phosphate and bind with high affinity to the phosphomannosyl receptor of human fibroblasts. In this study, we have partially characterized the Asn-linked oligosaccharide units present on these enzymes. [3H]Mannose-labeled alpha-D-mannosidase, beta-D-glucosidase, and beta-D-N-acetylglucosaminidase were purified from the spent growth medium of strain AX3 and glycopeptides were prepared by pronase digestion. Approximately 75% of the glycopeptides contained sulfate residues. These could be removed by solvolysis without degrading the underlying oligosaccharide. Following solvolysis (but not before), the oligosaccharides could be released by endo-beta-N-acetylglucosaminidase H, indicating the presence of high mannose-type units. Greater than 85% of the oligosaccharides contained one or two mannose 6-phosphate residues in the form of an unusual acid-stable phosphodiester. About 3% of the oligosaccharides contained phosphomonoesters and only 6% were neutral species. The major neutral oligosaccharide eluted in the position of Man9GlcNAc when analyzed by high performance liquid chromatography whereas the minor species appeared to be 1-2 residues larger. Acetolysis of the major phosphorylated fractions revealed that molecules with a single mannose 6-phosphate contained the phosphomannosyl residue on the branch linked alpha 1,6 to the beta-linked mannose whereas molecules with two phosphomannosyl residues had the residues on this branch as well as the branch linked alpha 1,3 to the beta-linked mannose. The mechanism of mannose phosphorylation in the slime mold must differ from that of mammalian cells since the phosphomannosyl residues are present as acid-resistant phosphodiesters rather than acid-labile phosphodiesters.

The recessive mutation, mod A, in the Dictyostelium discoideum strain M31 results in an alteration in the post-translational modification of lysosomal enzymes. We now report studies which indicate that mod A is deficient in glucosidase II, an enzyme which is involved in the processing of asparagine-linked oligosaccharides. [2-3H]Mannose-labeled glycopeptides were prepared from three purified mod A lysosomal enzymes and compared to the equivalent glycopeptides from parental enzymes. The mod A glycopeptides were deficient in high mannose oligosaccharides containing two phosphomannosyl residues and accumulated oligosaccharides with one phosphomannosyl residue. The phosphate was present in the form of an acid-stable phosphodiester in both instances. There was also an increase in the amount of nonphosphorylated high mannose oligosaccharides mod A and these were larger than the corresponding material from the parental enzymes. In addition, the nonphosphorylated oligosaccharides were only partially degraded by alpha-mannosidase, indicating the presence of a blocking moiety. In vitro enzyme assays demonstrated that the mod A cells cannot remove the inner 1 leads to 3-linked glucose from a glucosylated high mannose oligosaccharide. The cells are also deficient in membrane-bound neutral p-nitrophenyl-alpha-D-glucosidase activity. This activity has been attributed to glucosidase II in other systems. Removal of the outer 1 leads to 2-linked glucose from Glc3Man9Glc-NAc2 is normal, demonstrating the presence of glucosidase I activity. We conclude from these data that M31 cells are deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the glucosylated oligosaccharides of newly glycosylated proteins. This defect can explain the mod A phenotype and is proposed to be the primary genetic defect in these cells.