The Journal of Experimental Medicine

Dorsch, Marion; Fan, Pang-Dian; Danial, Nika N.; Rothman, Paul B.; Goff, Stephen P.

doi: 10.1084/jem.186.12.1947pmid: 9396763

Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor. Footnotes M. Dorsch is a fellow of the Howard Hughes Medical Institute. S.P. Goff is a Howard Hughes Medical Institute investigator. 1 Abbreviations used in this paper: EMSA, electrophoretic mobility-shift assay; MAPK, mitogen-activated protein kinase; MPLV, myeloproliferative leukemia virus; PI, phosphatidylinositol; STAT, signal transducers and activators of transcription; TPO, thrombopoietin. Submitted: 13 June 1997 Revision received 5 September 1997

Arase, Noriko; Arase, Hisashi; Park, Seung Yong; Ohno, Hiroshi; Ra, Chisei; Saito, Takashi

doi: 10.1084/jem.186.12.1957pmid: 9396764

Natural killer (NK) cells exhibit cytotoxicity against variety of tumor cells and virus-infected cells without prior sensitization and represent unique lymphocytes involved in primary host defense. NKR-P1 is thought to be one of NK receptors mediating activation signals because cross-linking of NKR-P1 activates NK cells to exhibit cytotoxicity and IFN-γ production. However, molecular mechanism of NK cell activation via NKR-P1 is not well elucidated. In this study, we analyzed the cell surface complex associated with NKR-P1 on NK cells and found that NKR-P1 associates with the FcRγ chain which is an essential component of Fc receptors for IgG and IgE. The association between FcRγ and NKR-P1 is independent of Fc receptor complexes. Furthermore, NK cells from FcRγ-deficient mice did not show cytotoxicity or IFN-γ production upon NKR-P1 cross-linking. Similarly, NK1.1 + T cells from FcRγ-deficient mice did not produce IFN-γ upon NKR-P1 crosslinking. These findings demonstrate that the FcRγ chain plays an important role in activation of NK cells via the NKR-P1 molecule. Footnotes The first two authors contributed equally to this work. 1 Abbreviations used in this paper: γ −/− , FcRγ-deficient; ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibition motif; KIR, killer inhibitory receptors. Submitted: 28 July 1997 Revision received 3 October 1997

Brumbaugh, Kathryn M.; Binstadt, Bryce A.; Billadeau, Daniel D.; Schoon, Renee A.; Dick, Christopher J.; Ten, Rosa M.; Leibson, Paul J.

doi: 10.1084/jem.186.12.1965pmid: 9396765

Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70–deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets. Footnotes This research was supported by the Mayo Foundation and by grant CA-47752 from the National Institutes of Health. 1 Abbreviations used in this paper: ADCC, antibody-dependent cell-mediated cytotoxicity; ITAM, immunoreceptor tyrosine-based activation motif; KAR, killer cell activating receptor; KARAP, KAR-associated protein; KIR, killer cell inhibitory receptor; PTK, protein tyrosine kinase. Kathryn M. Brumbaugh and Bryce A. Binstadt contributed equally to this work. Submitted: 7 August 1997 Revision received 9 October 1997

Chen, Gao; Ray, Reena; Dubik, Don; Shi, Lianfa; Cizeau, Jeannick; Bleackley, R. Chris; Saxena, Satya; Gietz, R. Dan; Greenberg, Arnold H.

doi: 10.1084/jem.186.12.1975pmid: 9396766

Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3 163 ) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3 163 mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3 163 protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression. Footnotes 1 Abbreviations used in this paper: α-MEM, α-minimal essential medium; 3AT, 3-amino-1,2,4-triazole. Submitted: 15 May 1997 Revision received 3 September 1997

Yu, Qin; Toole, Bryan P.; Stamenkovic, Ivan

doi: 10.1084/jem.186.12.1985pmid: 9396767

To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21–28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier. Footnotes 1 Abbreviations used in this paper: bPG, biotinylated proteoglycan; CMFDA, 5-chloromethyl-fluorescein diacetate; ECM, extracellular matrix; FBS, fetal bovine serum; FL-HA, fluorescein-labeled hyaluronan; HA, hyaluronan; ICAM, intracellular adhesion molecule; RT, reverse transcriptase; TUNEL, Tdt-mediated dUTP-biotin nick end labeling. Submitted: 3 June 1997 Revision received 15 September 1997

Matsumoto, Mitsuru; Fu, Yang-Xin; Molina, Hector; Huang, Guangming; Kim, Jinho; Thomas, Dori A.; Nahm, Moon H.; Chaplin, David D.

doi: 10.1084/jem.186.12.1997pmid: 9396768

In mice deficient in either lymphotoxin α (LT-α) or type I tumor necrosis factor receptor (TNFR-I), organized clusters of follicular dendritic cells (FDC) and germinal centers (GC) are absent from the spleen. We investigated the role of LT-α and TNFR-I in the establishment of spleen FDC and GC structure by using reciprocal bone marrow (BM) transfer. When LT-α–deficient mice were reconstituted with wild-type BM, FDC organization and the ability to form GC were restored, indicating that the LT-α–expressing cells required to establish organized FDC are derived from BM. The role of LT-α in establishing organized FDC structure was further investigated by the transfer of complement receptor 1 and 2 (CR1/2)–deficient BM cells into LT-α–deficient mice. Organized FDC were identified with both the FDC-M1 and anti-CR1 monoclonal antibodies in these BM-chimeric mice, indicating that these cells were derived from the LT-α–deficient recipient. Thus, expression of LT-α in the BM-derived cells, but not in the non–BM-derived cells, is required for the maturation of FDC from non-BM precursor cells. In contrast, when TNFR-I–deficient mice were reconstituted with wild-type BM, they showed no detectable FDC clusters or GC formation. This indicates that TNFR-I expression on non–BM-derived cellular components is necessary for the establishment of these lymphoid structures. TNFR-I–deficient BM was able to restore FDC organization and GC formation in LT-α–deficient mice, indicating that formation of these structures does not require TNFR-I expression on BM-derived cells. The data in this study demonstrate that FDC organization and GC formation are controlled by both LT-α–expressing BM-derived cells and by TNFR-I-expressing non–BM-derived cells. Footnotes D.D. Chaplin is an investigator of the Howard Hughes Medical Institute. Portions of this work were supported by grant AI34580 from the National Institutes of Health (D.D. Chaplin). 1 Abbreviations used in this paper: BM, bone marrow; FDC, follicular dendritic cell(s); GC, germinal center(s); IC, immune complex; LN, lymph node; LT, lymphotoxin; PNA, peanut agglutinin; TNFR, TNF receptor. Submitted: 23 June 1997 Revision received 8 October 1997

Akkaraju, Srinivas; Canaan, Karen; Goodnow, Christopher C.

doi: 10.1084/jem.186.12.2005pmid: 9396769

Graves' Disease results from the production of autoantibodies against receptors for thyroid stimulating hormone (TSH) on thyroid epithelial cells, and represents the prototype for numerous autoimmune diseases caused by autoantibodies that bind to organ-specific cell membrane antigens. To study how humoral tolerance is normally maintained to organ-specific membrane antigens, transgenic mice were generated selectively expressing membrane-bound hen egg lysozyme (mHEL) on the thyroid epithelium. In contrast to the deletion of autoreactive B cells triggered by systemic mHEL (Hartley, S.B., J. Crosbie, R. Brink, A.B. Kantor, A. Basten, and C.C. Goodnow. 1991. Nature. 353:765–769), selective expression of mHEL autoantigen on thyroid cells did not trigger elimination or inactivation of circulating HEL-reactive B cells. These results provide evidence that tolerance is not actively acquired to organ-specific antigens in the preimmune B cell repertoire, underscoring the importance of maintaining tolerance to such antigens by other mechanisms. The role of an intact endothelial barrier in sequestering organ-specific antigens from circulating preimmune B cells is discussed. Footnotes 1 Abbreviations used in this paper: cGG, chicken gamma globulin; HEL, hen egg lysozyme; mHEL, membrane-bound HEL; nPP, disodium p -nitrophenyl phosphate; pBS, Bluescript plasmid; rTg, rat thyroglobulin; sHEL, soluble HEL. Submitted: 1 July 1997 Revision received 14 October 1997

Turner, Martin; Gulbranson-Judge, Adam; Quinn, Marian E.; Walters, Alice E.; MacLennan, Ian C.M.; Tybulewicz, Victor L.J.

doi: 10.1084/jem.186.12.2013pmid: 9396770

The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool. Footnotes The work in London was supported by the Medical Research Council and the work in Birmingham was supported by a Medical Research Council program grant. 1 Abbreviations used in this paper: BCR, B cell antigen receptor; BLR1, Burkitt's lymphoma receptor 1; BrdU, 5′-bromo-2-deoxyuridine; ITAM, Immune receptor tyrosine-based activation motif. Submitted: 26 September 1997 Revision received 24 October 1997

Neumann, Harald; Schmidt, Hannes; Wilharm, Elke; Behrens, Lüder; Wekerle, Hartmut

doi: 10.1084/jem.186.12.2023pmid: 9396771

We explored expression and possible function of interferon-γ (IFN-γ) in cultured fetal (E15) rat dorsal root ganglion neurons combining whole cell patch-clamp electrophysiology with single cell reverse transcriptase polymerase chain reaction and confocal laser immunocytochemistry. Morphologically, we located IFN-γ protein in the cytoplasm of the neurons in culture as well as in situ during peri- and postnatal development. Transcripts for classic IFN-γ and for its receptor were determined in probes of cytoplasm sampled from individual cultured neurons, which had been identified by patch clamp electrophysiology. In addition, the cultured neurons expressed both chains of the IFN-γ receptor. Locally produced IFN-γ acts back on its cellular source. Phosphorylation and nuclear translocation of the IFN-inducible transcriptional factor STAT1 as well as IFN-γ–dependent expression of major histocompatibility complex class I molecules on the neuronal membrane were noted in untreated cultures. However, both processes were substantially blocked in the presence of antibodies neutralizing IFN-γ. Our findings indicate a role of IFN-γ in autocrine regulation of sensory neurons. Footnotes The project was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 391) and the European Community (CHRX-CT94-0670). 1 Abbreviations used in this paper: DRG, dorsal root ganglion; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; RT, reverse transcriptase. Submitted: 27 August 1997 Revision received 3 October 1997

Kessler, Benedikt; Hudrisier, Denis; Cerottini, Jean-Charles; Luescher, Immanuel F.

doi: 10.1084/jem.186.12.2033pmid: 9396772

Using H-2K d -restricted photoprobe-specific cytotoxic T lymphocyte (CTL) clones, which permit assessment of T cell receptor (TCR)-ligand interactions by TCR photoaffinity labeling, we observed that the efficiency of antigen recognition by CTL was critically dependent on the half-life of TCR-ligand complexes. We show here that antigen recognition by CTL is essentially determined by the frequency of serial TCR engagement, except for very rapid dissociations, which resulted in aberrant TCR signaling and antagonism. Thus agonists that were efficiently recognized exhibited rapid TCR–ligand complex dissociation, and hence a high frequency of serial TCR engagement, whereas the opposite was true for weak agonists. Surprisingly, these differences were largely accounted for by the coreceptor CD8. While it was known that CD8 substantially decreases TCR–ligand complex dissociation, we observed in this study that this effect varied considerably among ligand variants, indicating that epitope modifications can alter the CD8 contribution to TCR-ligand binding, and hence the efficiency of antigen recognition by CTL. Footnotes Dr. Denis Hudrisier was supported by an “Association Française pour la Recherche Thérapeutique” fellowship. Submitted: 7 July 1997 Revision received 10 September 1997