Multiple roles of Lyn kinase in myeloid cell signaling and function

Abstract

Lyn is an Src family kinase present in B lymphocytes and myeloid cells. In these cell types, Lyn establishes signaling thresholds by acting as both a positive and a negative modulator of a variety of signaling responses and effector functions. Lyn deficiency in mice results in the development of myeloproliferation and autoimmunity. The latter has been attributed to the hyper-reactivity of Lyn-deficient B cells due to the unique role of Lyn in downmodulating B-cell receptor activation, mainly through phosphorylation of inhibitory molecules and receptors. Myeloproliferation results, on the other hand, from the enhanced sensitivity of Lyn-deficient progenitors to a number of colony-stimulating factors (CSFs). The hyper-sensitivity to myeloid growth factors may also be secondary to poor inhibitory receptor phosphorylation, leading to impaired recruitment/activation of tyrosine phosphatases and reduced downmodulation of CSF signaling responses. Despite these observations, the overall role of Lyn in the modulation of myeloid cell effector functions is much less well understood, as often both positive and negative roles of this kinase have been reported. In this review, we discuss the current knowledge of the duplicitous nature of Lyn in the modulation of myeloid cell signaling and function.

Figure 1. Proposed model of the dual role of Lyn in the modulation of signaling pathways in myeloid cells

Lyn exerts its negative role in the modulation of signaling pathways in myeloid cells mainly by phosphorylating ITIM-containing inhibitory receptors, such as PIR-B and SIRPα thus recruiting inhibitory phosphatases, such as SHP1/2 or SHIP-1, which in turn downmodulate both proximal responses from Jak kinases and downstream responses at multiple sites distal to Ras. Additionally, Lyn can negatively modulate signaling responses in myeloid cells through the phosphorylation of the DOK family of cytoplasmic proteins that in turn recruit the Ras GTPase-activating protein (rasGAP) and SHIP-1 to down-modulate further signaling reactions. This inhibitory function of Lyn occurs especially in integrin, growth factor receptor and FcεRI signaling pathways. The mechanisms through which Lyn positively modulates signaling responses in myeloid cells are, on the other hand, less clear. Lyn can directly phosphorylate ITAM domains in various immunoreceptors associated signaling adapters (such as the FcRγ chain) leading to recruitment of Syk and activation of downstreamreactions. This positive regulatory function of Lyn occurs mainly in Fc signaling pathways. Lyn can also positively modulate signaling responses downstream to cytokine receptors, such as GM-CSF, IL-5, or chemokine receptors, but the mechanisms responsible for this phenomenon are not fully understood.

Figure 2. Lyn-deficient mice develop splenomegaly due to myeloproliferation and extra-medullary erythropoiesis

(A) Spleen weight of WT and lyn^−/− mice monitored over 60 weeks. (B) Splenic cells derived from WT and lyn^−/− mice stained with Wright/Giemsa. (C, D) Flow cytometric analysis of WT and lyn^−/− splenocytes from 6 and 28 week old mice. Percentages of Mac-1/Gr-1 double positive myeloid cells (C) or Ter-119 positive erythroid cells (D) are reported.

Figure 3. Myeloproliferation and loss of erythropoiesis in the bone marrow of Lyn-deficient mice

(A, B) Flow cytometric analysis of WT and lyn^−/− bone marrow from 6 and 28 week old mice. Percentages of Mac-1/Gr-1 double positive myeloid cells – mainly neutrophils – (A) or Ter-119 positive erythroid cells (B) are reported. (C) Paraffin embedded bone marrow sections from 28 week old WT and lyn^−/− mice probed with anti-TER-119 Ab.

Figure 4. Hyper-responsiveness of Lyn-deficient bone marrow cells to cytokine stimulation

(A) Cells derived from bone marrow and spleen obtained from WT, lyn^−/− and lyn^−/− rag^−/− (lacking mature B and T cells) mice at the indicated ages, were cultured in methylcellulose media supplemented with IL-3, IL-6, SCF and erythropoietin for 7 days and the resultant number of colony forming units were counted. Total CFU-C were calculated based on the total cell yield from each site (bone marrow or spleen). The experiments were repeated a minimum of three times in triplicate and averaged results are reported. (B) Equal numbers of bone marrow derived cells from WT and lyn^−/− mice were cultured with varying concentrations of GM-CSF or M-CSF for 7 days. Adherent cells number at the end of the culture period was determined using CyQuant fluorescent cell counting. The experiment was repeated a minimum of three times and the data shown are representative.

Figure 5. Lyn-deficient bone marrow derived macrophages manifest increased Erk1/2 activation and reduced inhibitory receptor phosphorylation following GM-CSF stimulation

(A) WT and lyn^−/− BMD macrophages were stimulated with 100ng/ml GM-CSF for varying lengths of time and Erk1/2 activation was determined by immunoblot (IB) using phospho-specific mAbs Lower panels show IBs with total Erk/1/2 to demonstrate equal loading. (B) WT and lyn^−/− BMD macrophages were stimulated with 100ng/ml GM-CSF for 15 minutes and cell lysates were immunoprecipitated (IP) with anti-Dok-1, anti-SIRPα and anti-PIR-B followed by anti-phosphotyrosine (anti-PY) immunoblotting (IB) to determine phosphorylation. Lower panels show IBs against the precipitated protein to demonstrate equal loading.

Figure 6. Lyn-deficient macrophages phagocytose apoptotic thymocytes normally

WT and lyn^−/− peritoneal macrophages were collected 5 days after IP injection with aged 3% thioglycollate and cultured overnight. Macrophages were exposed to 20-fold excess of apoptotic thymocytes, labeled with CellTracker green, for 30 min an examined by fluorescence microscopy, as described (108).