Ashley R. Dunn

Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease

Mice homozygous for a disruption at the Lyn locus display abnormalities associated with the B lymphocyte lineage and in mast cell function. Despite reduced numbers of recirculating B lymphocytes, Lyn-/- mice are immunoglobulin M (IgM) hyperglobulinemic. Immune responses to T-independent and T-dependent antigens are affected. Lyn-/- mice fail to mediate an allergic response to IgE cross-linking, indicating that activation of LYN plays an indispensable role in Fc epsilon RI signaling. Lyn-/- mice have circulating autoreactive antibodies, and many show severe glomerulonephritis caused by the deposition of IgG immune complexes in the kidney, a pathology reminiscent of systemic lupus erythematosus. Collectively, these results implicate LYN as having an indispensable role in immunoglobulin-mediated signaling, particularly in establishing B cell tolerance.

Mice with a null mutation of the TGFα gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation

Mice homozygous for a disrupted transforming growth factor alpha (TGF alpha) gene are healthy and fertile, although some older mice show evidence of corneal inflammation. In contrast with TGF alpha +/- and +/+ animals, TGF alpha -/- mice have a pronounced waviness of the coat. Histological examination of the skin from TGF alpha -/- mice reveals a dramatic derangement of hair follicles. Mice with a disrupted TGF alpha gene also have curly whiskers, first evident on the day of birth. The phenotype of TGF alpha -/- mice is remarkably similar to that of the mouse mutant waved-1 (wa-1). Offspring resulting from crosses between TGF alpha -/- and wa-1 mice display the curly whisker-coat phenotype, indicating that the basis of the wa-1 phenotype is a mutation in the TGF alpha gene. These observations suggest that TGF alpha plays a pivotal role in determining skin architecture and in regulating hair development.

POLYGENIC AUTOIMMUNE TRAITS : LYN, CD22, AND SHP-1 ARE LIMITING ELEMENTS OF A BIOCHEMICAL PATHWAY REGULATING BCR SIGNALING AND SELECTION

A B lymphocyte hyperactivity syndrome resembling systemic lupus erythematosus characterizes mice lacking the src-family kinase Lyn. Lyn is not required to initiate B cell antigen receptor (BCR) signaling but is an essential inhibitory component. lyn-/- B cells have a delayed but increased calcium flux and exaggerated negative selection responses in the presence of antigen and spontaneous hyperactivity in the absence of antigen. As in invertebrates, genetic effects of loci with only one functional allele can be used to analyze signaling networks in mice, demonstrating that negative regulation of the BCR is a complex quantitative trait in which Lyn, the coreceptor CD22, and the tyrosine phosphatase SHP-1 are each limiting elements. The biochemical basis of this complex trait involves a pathway requiring Lyn to phosphorylate CD22 and recruit SHP-1 to the CD22/BCR complex.

Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage

Transgenic mice carrying the murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene expressed from a retroviral promoter exhibit elevated levels of GM-CSF in the serum, urine, peritoneal cavity, and eye. The eyes of transgenic mice are opaque, contain accumulations of macrophages, and develop retinal damage. Similarly, lesions containing macrophages develop in striated muscle. The mice also display an accumulation of large, often multinucleate, activated macrophages in the peritoneal and pleural cavities. The transgene is transcribed in peritoneal cells, as well as in eyes and infiltrated striated muscle. A high proportion of transgenic mice die with muscle wasting when aged 2-4 months, possibly because of macrophage activation resulting from the high levels of GM-CSF.

gp130-mediated Signal Transduction in Embryonic Stem Cells Involves Activation of Jak and Ras/Mitogen-activated Protein Kinase Pathways

The leukemia inhibitory factor/interleukin 6 (LIF/IL6) family of cytokines promotes cell type-specific pleiotropic effects by engaging multimeric receptor complexes that share the common affinity converter/signal transducing subunit gp130. While the maintenance of embryonic stem (ES) cell self-renewal is an activity unique to this family of cytokines, the intracellular signaling events mediated by gp130 remain largely unknown. Here we show a rapid and transient increase in the specific activity of the Src-related kinase Hck as well as of the Janus kinases Jak1, Jak2, and Tyk2 following treatment of ES cells with LIF or a combination of IL6 plus a soluble form of the IL6 receptor. Within 2 min of stimulation, we also observed increased tyrosine phosphorylation of SHC, activation of the guanidine nucleotide exchange activity on p21ras, and an electrophoretic mobility shift of MAP kinase. Functional involvement of Hck and p21ras activation in gp130-mediated signaling is supported by the finding that the introduction of constitutively activated Hck or v-Ha-ras partially alleviates the requirement of ES cells for LIF to remain undifferentiated. In contrast, suppression of Jak1 in ES cells by antisense technology increased the amount of LIF required to retain their pluripotentiality. These results are consistent with the notion that gp130-mediated suppression of ES cell differentiation depends on signaling through at least two cascades, namely a p21ras-dependent pathway that possibly involves Hck, as well as a Jak kinase-dependent pathway.

Point mutations within a dimer interface homology domain of c-Mpl induce constitutive receptor activity and tumorigenicity.

c‐Mpl, a receptor for thrombopoietin (TPO), belongs to the haemopoietin/cytokine receptor superfamily, a group of cell surface molecules characterized by conserved sequence motifs within their ligand binding domains. A recurring mechanism for the activation of haemopoietin receptors is the formation of functional complexes by receptor subunit oligomerization. Within the growth hormone receptor, a cluster of extracellular amino acids forms a dimer interface domain that stabilizes ligand‐induced homodimers. This domain appears to be functionally conserved in the erythropoietin (EPO) receptor because substitution of cysteines for residues in the analogous region causes EPO‐independent receptor activation via disulfide‐linked homodimerization. This report identifies an homologous domain within the c‐Mpl receptor. The substitution of cysteine residues for specific amino acids in the dimer interface homology regions of c‐Mpl induced constitutive receptor activity. Factor‐dependent FDC‐P1 and Ba/F3 cells expressing the active receptor mutants no longer required exogenous factors and proliferated autonomously. The results imply that the normal process of TPO‐stimulated Mpl activation occurs through receptor homodimerization and is mediated by a conserved haemopoietin receptor dimer interface domain. Moreover, cells expressing activated mutant Mpl receptors were tumorigenic in transplanted mice. Thus, like v‐mpl, its viral counterpart, mutated forms of the cellular mpl gene also have oncogenic potential.

Gain- and Loss-of-Function Lyn Mutant Mice Define a Critical Inhibitory Role for Lyn in the Myeloid Lineage

To investigate the role of the Lyn kinase in establishing signaling thresholds in hematopoietic cells, a gain-of-function mutation analogous to the Src Y527F-activating mutation was introduced into the Lyn gene. Intriguingly, although Lyn is widely expressed within the hematopoietic system, these mice displayed no propensity toward hematological malignancy. By contrast, analysis of aging cohorts of both loss- and gain-of-function Lyn mutant mice revealed that Lyn(-/-) mice develop splenomegaly, increased numbers of myeloid progenitors, and monocyte/macrophage (M phi) tumors. Biochemical analysis of cells from these mutants revealed that Lyn is essential in establishing ITIM-dependent inhibitory signaling and for activation of specific protein tyrosine phosphatases within myeloid cells. Loss of such inhibitory signaling may predispose mice lacking this putative protooncogene to tumorigenesis.

Structure and expression of the mRNA for murine granulocyte-macrophage colony stimulating factor

A cDNA containing a virtually complete copy of the mRNA for the haemopoietic growth regulator, granulocyte‐macrophage colony stimulating factor (GM‐CSF), has been isolated from a murine T lymphocyte cDNA library. When a eukaryotic expression vector with this cDNA coupled to the SV40 late promoter was introduced into simian COS cells, significant quantities of GM‐CSF were secreted. Since all of the biological activities previously ascribed to highly purified GM‐CSF were exhibited in the COS cell‐derived GM‐CSF, all of these activities are intrinsic to the product of a single gene. There are two potential translational initiation codons in the GM‐CSF mRNA; the first is buried in the stem and the second located in the loop of a very stable hairpin structure. Expression studies using deletion derivatives of the cDNA indicated that the second AUG is able to initiate the translation and secretion of GM‐CSF. The amino acid sequence of the leader peptide is rather atypical for a secreted protein and we speculate that molecules which initiate at the first AUG might exist as integral membrane proteins whereas those initiating at the second are secreted.

Sustained Activation of Lyn Tyrosine Kinase In Vivo Leads to Autoimmunity

Genetic ablation of the Lyn tyrosine kinase has revealed unique inhibitory roles in B lymphocyte signaling. We now report the consequences of sustained activation of Lyn in vivo using a targeted gain-of-function mutation (Lynup/up mice). Lynup/up mice have reduced numbers of conventional B lymphocytes, down-regulated surface immunoglobulin M and costimulatory molecules, and elevated numbers of B1a B cells. Lynup/up B cells are characterized by the constitutive phosphorylation of negative regulators of B cell antigen receptor (BCR) signaling including CD22, SHP-1, and SHIP-1, and display attributes of lymphocytes rendered tolerant by constitutive engagement of the antigen receptor. However, exaggerated positive signaling is also apparent as evidenced by the constitutive phosphorylation of Syk and phospholipase Cγ2 in resting Lynup/up B cells. Similarly, Lynup/up B cells show a heightened calcium flux in response to BCR stimulation. Surprisingly, Lynup/up mice develop circulating autoreactive antibodies and lethal autoimmune glomerulonephritis, suggesting that enhanced positive signaling eventually overrides constitutive negative signaling. These studies highlight the difficulty in maintaining tolerance in the face of chronic stimulation and emphasize the pivotal role of Lyn in B cell signaling.

The structure and expression of the murine gene encoding granulocyte-macrophage colony stimulating factor: evidence for utilisation of alternative promoters.

Two overlapping genomic clones containing the murine granulocyte‐macrophage colony stimulating factor (GM‐CSF) gene have been isolated. On the basis of transfection experiments, we have established that a 9‐kb BamHI fragment from one of these recombinants encodes biologically active GM‐CSF. As deduced from nucleotide sequence analysis, the GM‐CSF gene comprises four exons encompassing 2.5 kb of genomic DNA. Primer extension analysis of GM‐CSF mRNA identifies a transcriptional initiation site 35 bp upstream of a single translational initiation codon in‐frame with the GM‐CSF coding sequences and 28 bp downstream of a TATA promoter consensus sequence. Pre‐GM‐CSF molecules encoded by mRNAs originating from this promoter would include a hydrophobic leader sequence typical for a secreted protein. Intriguingly, sequences present at the 5′ end of a GM‐CSF cDNA clone previously isolated in our laboratory are not contained within either of the genomic clones and must therefore be transcribed from a promoter located at least 10 kb 5′ of the main body of the gene. mRNAs transcribed from this alternative upstream promoter possess an additional initiating codon and potentially encode a pre‐GM‐CSF polypeptide with an atypical NH2‐terminal leader peptide. Comparison of the nucleotide sequence of the GM‐CSF gene with that of other haemopoietic growth factor genes has revealed a common decanucleotide (5′‐GPuGPuTTPyCAPy‐3′) within their respective 5′‐flanking regions which may be involved in their co‐ordinate regulation.