Takuya Katagiri

An Established MRL/Mp-lpr/lpr Cell Line with Null Cell Properties Produces a B Cell Differentiation Factor(s) That Promotes Anti-Single-Stranded DNA Antibody Production in MRL Spleen Cell Culture

The cell line established from the lymph node cells of an MRL/lpr mouse, was found to have null cell properties in that it lacked Thy-1, Lyt-1 and Lyt-2 as well as sIg, and continued to grow in the absence of exogeneously added lymphokines such as IL-2 and IL-3. Interestingly, this cell line (KML1) or a soluble factor(s) produced by it promoted anti-ssDNA antibody production in cultures of MRL/lpr spleen cells. The factor did not induce cell proliferation. Therefore, it is concluded that the cell line KML1 produced at least a B-cell differentiation factor, but not IL-2 or IL-3 as far as detected with the respective lymphokine-dependent cell lines.

Ubiquitin-proteasome system is involved in induction of LFA-1/ICAM-1-dependent adhesion of HL-60 cells.

Membrane‐permeable proteasome inhibitors, lactacystin (LC) and N‐acetyl‐Leu‐Leu‐norleucinal (ALLN), but not calpain inhibitor Z‐Leuleucinal (ZLL), prevented LFA‐1/ICAM‐1‐dependent cellular adhesion of TPA‐stimulated HL‐60 cells. These proteasome inhibitors affected neither the induction of monocytic differentiation nor the accompanying protein‐tyrosine phosphorylation. They suppressed the increase in the avidity of LFA‐1 to ICAM‐1 without changing the expression of these molecules. Immunoblotting using monoclonal antibody FK‐1, which reacts specifically with polyubiquitinated proteins, demonstrated that the proteasome inhibitors caused the drastic accumulation of the polyubiquitinated proteins in the membrane fraction of TPA‐treated HL‐60 cells. This indicates that accompanying activation of LFA‐1, TPA induces the polyubiquitination of the membrane proteins, which are rapidly degraded by proteasomes. These data taken together show that proteolysis mediated by the ubiquitin‐proteasome system is a prerequisite for the induction of LFA‐1‐dependent adhesion of HL‐60 cells. J. Leukoc. Biol. 65: 778–785; 1999.

Modulation of monocytic differentiation of HL-60 cells by inhibitors of protein tyrosine kinases

We showed previously that the expressions of various src family protein tyrosine kinases (PTKs) were induced independently during the monocytic differentiation of HL-60 cells. The role of PTKs was further assessed in the present study by investigating the effects of PTK inhibitors on the differentiation. It was demonstrated that PTK inhibitors such as genistein and herbimycin A modulated monocytic differentiation of HL-60 cells; they inhibited the differentiation induced by TPA, while promoting that induced by vitamin D3 (D3). Immunoblotting analysis of protein molecules which had been phosphorylated on their tyrosine residues demonstrated that TPA induced phosphorylation of certain molecules different from those induced by D3 in HL-60 cells. PTK inhibitors blocked the phosphorylation and modulated differentiation driven by the inducers. These data suggest that PTKs are involved both promotively and suppressively in signaling events that induce monocytic differentiation of HL-60 cells.

Characterization of lymphoproliferation induced by interactions between Iprcg and gld genes

The lprcg gene is the novel mutation at the lpr locus characterized by its complementary to the gld gene in induction of lymphoproliferation in the mouse. Because of the potential usefulness of mice with this mutation in studies on the interrelationship between lpr and gld, we were urged to characterize the lymphoproliferative disease developing in (CBA/K1Jms-lprcg/lprcg x C3H/HeJ-gld/gld) F1 hybrid (lprcg-gld) mice. Despite the milder lymphadenopathy in the lprcg-gld mice, the expanding lymph node cells showed the same surface marker pattern as that in C3H/HeJ-lpr/lpr, C3H/HeJ-gld/gld, and CBA/K1Jms-lprcg/lprcg mice, characterized by the positivity for Thy-1, B220, Ly-6, and Ly-24, and the negativity for L3T4, Lyt-2 (hence designated double-negative cells), and sIg. Furthermore, these cells proved to be of a T-cell lineage based on the rearrangement of the TCR beta-chain gene, the same as the already known double-negative cells. Noticeably, in lprcg-gld mice, serum IgG and autoantibodies of the IgG class were not elevated at an early age but were slightly elevated at an advanced age despite early elevation of the serum IgM and IgM autoantibodies. These results suggest that the lymphoproliferative mice carrying lprcg and gld genes in a heterozygous state will serve as a new tool for inquiring into the interrelationship among lpr, gld, and lprcg.

Interleukin-2 Responses of MRL/lpr Mouse Splenocytes and Lymph Node Cells Induced by TPA and A23187

Treatment of splenocytes and lymph node cells of 5 month-old MRL/lpr mice with TPA induced IL-2-dependent proliferation of the cells in the presence of CA++. The induced response was inhibited completely by a monoclonal antibody to IL-2 receptor. The combination of TPA and A23187 in the lpr cells induced both proliferation and production of IL-2 in a Ca++-dependent fashion. The proliferative response of the lpr cells was equivalent to that of congenic (MRL/+/+) or normal cells, but the quantity of IL-2 secreted from the lpr cells was significantly less than that of the controls. Actinomycin D, but not mitomycin C, blocked IL-2 secretion from the treated lpr cells indicating de novo synthesis of IL-2 mRNA in the cells. Thus the lpr lymphocytes can be activated to proliferate in response to IL-2, yet they do not secrete IL-2 at a normal level even if activation signals are transmitted into the cells.

Polyclonal Antibody Synthesis by Bone Marrow Cells of New Zealand Black Mice

The activity of polyclonal antibody synthesis (PAS) of bone marrow cells (BMC) was assessed by enumerating anti-trinitrophenyl (TNP) plaque-forming cells (PFC) or immunoglobulin-secreting cells (IgSC) generated in the spleen of BMC-reconstituted syngeneic or H-2-compatible hosts. BMC of NZB mice generated large numbers of anti-TNP PFC or IgSC as compared with those of C57BL/6 mice or H-2-compatible, nonautoimmune strains of mice (BALB/c, DBA/2 and B10 . D2 mice). Causes for the enhanced PAS activity were explored. Susceptibility to known B cell mitogens was examined and it was shown that spleen cells of the recipients of NZB BMC were refractory to the mitogens while PAS was apparent and acquired responsiveness after 15 days as those from nonautoimmune strains of mice. Furthermore, environmental factor(s) did not seem to be responsible for the enhanced PAS, since BMC from nonautoimmune BALB/c mice showed little PAS activity when transferred into heavily irradiated NZB mice and those of NZB mice showed enhanced PAS activity when transferred into B10 . D2 or BALB/c mice. It seems most likely that enhanced PAS activity of BMC of NZB mice is a genetically inherent phenomenon. PAS activity of BMC and spleen cells of NZB mice increased significantly with increasing age of mice.

Anti-Apoptotic Role of Protein-Tyrosine Kinases During Granulocytic Differentiation of HL-60 Cells

The human promyelocytic leukemia cell line HL-60 can be induced to differentiate towards neutrophils and subsequently die via apoptosis in vitro In this paper, we investigated the roles of protein tyrosine kinases (PTKs) in retinoic acid (RA)-induced granulocytic differentiation of HL-60 cells. Accompanying the RA-induced differentiation, Lyn and Fgr PTKs were phosphorylated on their tyrosine residues and their activities were induced. The degree of both activities and tyrosinephosphorylation of these PTKs was reduced to be minimal at day 5 when the HL-60 cells start to be dead by apoptosis. The inhibitors of PTKs, herbimycin A and genistine, were demonstrated to cause premature cell death of HL-60 cells in the presence of RA. The death was the consequence of an apoptotic process. The RA-treated HL-60 cells, when incubated with specific c-lyn or c-fgr antisense oligodeoxy-nucleotide, also underwent premature death. These data implicate that Lyn and Fgr PTKs prevent programmed cell death to promote granulocytic differentiation of HL-60 cells.