Nicholas M. Gough

Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor.

Two cDNA clones encoding a receptor for human granulocyte‐macrophage colony‐stimulating factor (hGM‐CSF‐R) were isolated by expression screening of a library made from human placental mRNA. Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodinated hGM‐CSF using a sensitive microscopic autoradiographic approach. The cloned GM‐CSF‐R precursor is a 400 amino acid polypeptide (Mr 45,000) with a single transmembrane domain, a glycosylated extracellular domain and a short (54 amino acids) intracytoplasmic tail. It does not contain a tyrosine kinase domain nor show homology with members of the immunoglobulin super gene family, but does show some significant sequence homologies with receptors for several other haemopoietic growth factors, including those for interleukin‐6, erythropoietin and interleukin‐2 (beta‐chain) and also to the prolactin receptor. When transfected into COS cells the cloned cDNA directed the expression of a GM‐CSF‐R showing a single class of affinity (KD = 2(‐8) nM) and specificity for human GM‐CSF but not interleukin‐3. Messenger RNA coding for this receptor was detected in a variety of haemopoietic cells known to display hGM‐CSF binding, and cross‐linking experiments revealed a similar size for the glycosylated receptors in transfected COS and haemopoietic cells.

Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF).

Leukaemia inhibitory factor (LIF) can induce macrophage differentiation in M1 murine myeloid leukaemic cells and suppress their proliferation in vitro. It does not stimulate the proliferation of normal progenitor cells and is apparently distinct from known colony‐stimulating factors. We have used oligo‐nucleotides complementary to partial amino acid sequence of LIF to isolate a LIF clone from a T lymphocyte cDNA library. When this cDNA was coupled to a yeast expression vector (YEpsec1) and introduced into yeast cells, a molecule with the biological properties characteristic of native LIF was secreted into the growth medium. The amino acid sequence of LIF established it to be a unique molecular entity, distinct from the other known haemopoietic growth factors. Since LIF is encoded by a unique gene, two biochemically separable forms of LIF probably represent post‐transcriptional or posttranslational variants of the same gene product. In contrast to several other haemopoietic regulators, the 0.8‐ to 1‐kb LIF mRNA was expressed constitutively in two murine T lymphocyte cell lines examined, and its abundance was not enhanced by stimulation with concanavalin A. Cloning, sequencing and expressing LIF has resolved several discrepancies in the literature concerning the identity of factors capable of inducing differentiation of murine myeloid leukaemic cells in vitro.

Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells

An extremely simple procedure for preparing cytoplasmic RNA from small numbers of cells is described. Cells are lysed with the detergent NP-40 and efficient extraction of protein from the postnuclear cytoplasmic lysate is ensured by denaturation with sodium dodecyl sulfate and urea. This procedure is suitable for preparing RNA from many cell types. All procedures have been scaled down to be performed in 1.5-ml microfuge tubes and thus RNA may be prepared from small numbers of cells. The procedure is extremely rapid and RNA is ready for Northern gel analysis in less than 30 min. Because so few steps are involved, RNA recovery is quantitative.

Cloning of a murine IL-11 receptor alpha-chain; requirement for gp130 for high affinity binding and signal transduction.

An adult mouse liver cDNA library was screened with oligonucleotides corresponding to the conserved WSXWS motif of the haemopoietin receptor family. Using this method, cDNA clones encoding a novel receptor were isolated. The new receptor, named NR1, was most similar in sequence and predicted structure to the alpha‐chain of the IL‐6 receptor and mRNA was expressed in the 3T3‐L1 pre‐adipocytic cell line and in a range of primary tissues. Expression of NR1 in the factor‐dependent haemopoietic cell line Ba/F3 resulted in the generation of low affinity receptors for IL‐11 (Kd approximately 10 nM). The capacity to bind IL‐11 with high affinity (Kd = 300‐800 pM) appeared to require coexpression of both NR1 and gp130, the common subunit of the IL‐6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) receptors. The expression of both NR1 and gp130 was also necessary for Ba/F3 cells to proliferate and M1 cells to undergo macrophage differentiation in response to IL‐11.

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.

Heterogeneity in Lymphokine Profiles of CD4+ and CD8+ T Cells and Clones Activated in vivo and in vitro

Analysis of lymphokine mRNA expression and protein secretion by about 100 short-term alloreactive T-cell clones revealed marked heterogeneity in the combinations of lymphokines synthesized. This finding argues against a simple model in which T cells express either an unrestricted (Th0) or a restricted (Th1 or Th2) lymphokine profile. Lymphokine titers appeared to be normally distributed, with the percentage of positive clones for any one product determined by the threshold of detection. Accordingly, the observation that CD4+ clones on average produced higher titers of most lymphokines than CD8+ clones indicated that apparent differences between the lymphokine profiles of these two subsets were quantitative rather than qualitative. Patterns of lymphokine gene expression detected in whole tissues or by analysis of single cells and clones were markedly influenced by in vivo priming. Relative levels of expression of IL-4, IFN-gamma and GM-CSF in lymphoid tissues differed in mice undergoing a GvHR or following contact sensitization with OX or immunization with KLH in adjuvant. Consistent with the finding that IL-4 was the major lymphokine mRNA detected in lymph nodes of KLH-primed mice, most short-term KLH-specific clones derived from such mice also expressed IL-4. A similar approach to the detection of lymphokine-secreting T-cell precursors activated late in L. major infection showed that most clones from the L. major-resistant strain, C57BL/6, secreted IFN-gamma without IL-4 whereas most clones from the susceptible strain, BALB/c, secreted IL-4 without IFN-gamma. Differences were also noted in anti-CD3-induced IL-3 production at the single-cell level between CD8+ cells activated in the GvHR or against a tumor allograft. Con A-induced, filler cell-dependent cloning of CD4+ T cells from unprimed mice gave rise both to IFN-gamma-producing and to IL-4-producing clones. A requirement for an undefined, filler cell-dependent signal for development of IL-4-secreting clones was suggested by the finding that clones of normal CD4+ and CD8+ T cells activated in an anti-CD3-induced, filler cell-free system exclusively produced IFN-gamma and IL-3 without detectable IL-4 or IL-6. With a view to developing a single-cell approach to the analysis of lymphokine profiles of in vivo-activated T cells, sensitive assays for IL-3 and other lymphokines were used to measure secreting cells activated in the GvHR or against a tumor allograft.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Characterization of cytokine production by the metrial gland and granulated metrial gland cells

The metrial gland and its population of bone marrow-derived, large, granulated, lymphocyte-like cells, termed granulated metrial gland (GMG) cells, are consistent but poorly understood, decidua-associated features of pregnancy in the mouse and other species. Decidua, a complex maternal tissue, is thought to be a source of cytokines important for placental development. Thus, it is important to determine if lymphokine or cytokine production is among the activities of the metrial gland and GMG cells. Media conditioned by culture of either metrial gland explants or migrating GMG cells were evaluated for various cytokine activities. At least four activities were present: CSF-1, IL-1, a factor promoting proliferation of DA-1 cells that was not GM-CSF, IL-3 or erythropoietin and an activity cytotoxic to the CSF-1-dependent macrophage cell line 5/10.14. CSF-1 and IL-1 appeared to be products of the GMG cells. Cytokines not present at detectable levels included IL-2, IL-4, TNF-alpha and TGF-beta. Qualitatively, the cytokine profiles remained constant throughout days 8-16 of gestation. mRNA from migratory GMG cells was isolated and assayed for eleven cytokine mRNAs by polymerase chain reaction-based amplification of cDNA synthesized from mRNA. GMG cell RNA contained transcripts for LIF and CSF-1 but did not contain transcripts for GM-CSF, G-CSF, IL-2, IL-3, IL-4, IL-6, IL-7, IFN-gamma or TNF-alpha. TGF-beta transcripts were detected in occasional samples at very low levels. Since GMG cells are highly mobile cells that migrate throughout the placenta and into trophoblast-lined maternal blood spaces, their function in pregnancy may involve the delivery of very localized differentiation or growth regulatory signals to the developing fetal trophoblast and placenta.

Leukaemia inhibitory factor and interleukin 6 are expressed at very low levels in the normal adult mouse and are induced by inflammation

We have assessed the limitations of the polymerase chain reaction (PCR) as a semiquantitative technique for assessing very low level gene expression. Using PCR, the in vivo expression patterns of the cytokines Leukaemia Inhibitory Factor (LIF) and Interleukin 6 (IL-6) in the normal adult mouse, have been examined. We show that both LIF and IL-6 mRNA are constitutively expressed, albeit at extremely low levels, in most tissues. While it is unclear whether this low level of expression is of biological significance, it is possible that it reflects a local mode of action of these potent polyfunctional molecules. Lipopolysaccharide, the bacterial cell wall product responsible for endotoxic shock, when administered in vivo, was capable of inducing the expression of both LIF and IL-6 in all of the tissues examined. In addition, LIF and IL-6 expression was induced in lung tissue by in vitro culturing in serum-free media. This induction of LIF and IL-6, by LPS and culturing, may reflect the role of these molecules as mediators of the acute phase response to tissue damage.

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF‐R), while mouse LIF (mLIF) binds only to mouse LIF‐R. Moreover, hLIF binds with higher affinity to the mLIF‐R than does mLIF. In order to define the regions of the hLIF molecule responsible for species‐specific interaction with the hLIF‐R and for the unusual high‐affinity binding to the mLIF‐R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three‐dimensional structure. Since all chimeras retained full biological activity and receptor‐binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF‐R were unaltered by inter‐species domain swapping. Consequently, the site on the hLIF molecule that confers species‐specific binding to the hLIF‐R and higher affinity binding to the mLIF‐R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF‐R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins.