Transferrin Receptor 1 Facilitates Poliovirus Permeation of Mouse Brain Capillary Endothelial Cells

As a possible route for invasion of the CNS, circulating poliovirus (PV) in the blood is believed to traverse the blood-brain barrier (BBB), resulting in paralytic poliomyelitis. However, the underlying mechanism is poorly understood. In this study, we demonstrated that mouse transferrin receptor 1 (mTfR1) is responsible for PV attachment to the cell surface, allowing invasion into the CNS via the BBB. PV interacts with the apical domain of mTfR1 on mouse brain capillary endothelial cells (MBEC4) in a dose-dependent manner via its capsid protein (VP1). We found that F-G, G-H, and H-I loops in VP1 are important for this binding. However, C-D, D-E, and E-F loops in VP1-fused Venus proteins efficiently penetrate MBEC4 cells. These results imply that the VP1 functional domain responsible for cell attachment is different from that involved in viral permeation of the brain capillary endothelium. We observed that co-treatment of MBEC4 cells with excess PV particles but not dextran resulted in blockage of transferrin transport into cells. Using the Transwell in vitro BBB model, transferrin co-treatment inhibited permeation of PV into MBEC4 cells and delayed further viral permeation via mTfR1 knockdown. With mTfR1 as a positive mediator of PV-host cell attachment and PV permeation of MBEC4 cells, our results indicate a novel role of TfR1 as a cellular receptor for human PV receptor/CD155-independent PV invasion of the CNS.     

Specific neonatally induced tolerance to Mls locus determinants

Neonatal injection of CBA/HT6T6 (H-2k, Mlsb) mice with adult, Mls-incompatible (CBA/J [H-2k, Mlsd] X CBA/HT6T6)F1 spleen cells results in the abrogation of cell proliferation and interleukin 2 (IL 2) production in bulk mixed lymphocyte cultures, when spleen cells from the inoculated mice are tested at 6 to 8 wk of age with stimulator cells expressing the Mlsd of the tolerizing inoculum. In limiting dilution assays, this tolerant state was manifested in a 25- to 550-fold (280-fold average) decrease in the frequency of precursors of Mlsd-responsive IL 2-producing T cells. Tolerance was specific in that the frequencies of precursors of IL 2-producing cells responding to Con A, allogeneic H-2d, and self-Ia were not affected. The observed low frequency of Mls-responsive cells was due neither to extensive chimerism resulting in the dilution of Mlsd-responsive cells by the nonresponsive F1 cells of the inoculum, nor to the action of suppressor cells. These findings indicate that neonatal injection of Mls-incompatible spleen cells produces a state of specific tolerance by a clonal deletion or inactivation mechanism. This specific tolerance supports the view that 1) the Mls locus encodes or regulates the expression of defined alloantigenic determinants and 2) Mls-incompatible responder mice have specific receptors for Mls determinants on clonally distributed IL 2-producing responder T cells.     

Release of histamine and arachidonate from mouse mast cells induced by glycosylation-enhancing factor and bradykinin

Stimulation of normal rat splenic T cells with pertussigen (lymphocytosis-promoting factor from Bordetella pertussis) resulted in the release of a soluble factor that enhanced the assembly of N-linked oligosaccharides to IgE-binding factors during their biosynthesis. The glycosylation-enhancing factor (GEF) is a kallikrein-like enzyme and is purified by absorption to p-aminobenzamidine-Agarose followed by elution with benzamidine. Incubation of normal mouse mast cells with affinity-purified GEF or bradykinin, a product of cleavage of kininogen by kallikrein, resulted in the release of histamine and arachidonate from the cells. Passive sensitization of mast cells with mouse IgE antibody, followed by pretreatment of the cells with a suboptimal concentration of GEF, resulted in an enhancement of antigen-induced histamine release. It was found that GEF and bradykinin induced the same biochemical events in mast cells as those induced by bridging of IgE receptors. Both GEF and bradykinin induced phospholipid methylation and an increase in intracellular cyclic AMP (cAMP). Incorporation of 3H-methyl groups into phospholipids and intracellular cAMP levels both reached a maximum 30 sec after challenge with GEF or bradykinin, and then declined to base-line levels within 2 to 3 min. These biochemical events were followed by 45Ca influx and histamine release; 45Ca uptake reached a plateau value at 2 min, and histamine release reached a maximum at 5 to 8 min. The initial rise in cAMP induced by GEF (or bradykinin) was not inhibited by indomethacin, indicating that the activation of adenylate cyclase is not the result of prostaglandin synthesis. In both IgE-mediated and GEF-induced histamine release, inhibitors of methyltransferases, such as 3-deaza adenosine and L-homocysteine thiolactone, inhibited not only phospholipid methylation but also the cAMP rise and subsequent Ca2+ uptake and histamine release. The results indicate that GEF induces activation of methyltransferases and that phospholipid methylation is involved in the cAMP rise, Ca2+ uptake, and histamine release. The induction of the same biochemical events in the same sequence by bridging of IgE receptors and by GEF (bradykinin) supports the hypothesis that receptor bridging induces the activation of serine protease(s) and cleavage products of this enzyme in turn activate methyltransferases in mast cells.     

Morphologic and immunologic characterization of human basophils developed in cultures of cord blood mononuclear cells

Selective growth of human basophilic granulocytes was obtained in suspension cultures of mononuclear cells from umbilical cord blood. Approximately 50 to 80% of nonadherent cells recovered from 2- to 3-wk-old cultures contained metachromatic granules, and these cells were identified as human basophilic granulocytes by electron microscopy. Histamine content of cultured human basophils was comparable to that in peripheral blood basophils. Cultured basophils bear 2.7 to 3.7 X 10(5) IgE receptors per cell that bind both human IgE and rodent IgE with comparable affinity. Average equilibrium constants of the receptors for human IgE and mouse IgE were 2.56 +/- 0.88 X 10(9) M-1 and 1.85 +/- 0.86 X 10(9) M-1, respectively. The cell-surface component of the IgE receptors on cultured basophils has a m.w. of 64,000. Cultured basophils could be passively sensitized with human IgE and mouse IgE monoclonal antibody, and sensitized basophils released characteristic cytoplasmic granules and both histamine and arachidonate upon challenge with either anti-human IgE or antigen. Incubation of cultured basophils with ionophore A23187 or F-Met-Leu-Phe resulted in histamine release. However, compound 48/80 failed to induce histamine release from the cells.     

Participation of IL-4 in the formation of IgD-binding factors by antigen-primed mouse spleen cells

BALB/c mouse spleen cells primed with either keyhole limpet hemocyanin or DNP-keyhole limpet hemocyanin formed not only IgG-binding factors (BF) and IgE-BF but also IgD-BF upon antigenic stimulation. Analysis of cellular mechanisms involved in the formation of Ig-BF by antigenic stimulation revealed that Ag-primed Th cells released lymphokines upon antigenic stimulation, and that the lymphokine(s) in turn stimulates unprimed T cells to form Ig-BF. Normal unprimed lymphocytes formed IgD-BF upon incubation with culture supernatants of Ag-stimulated spleen cells. The formation of IgD-BF induced by the culture supernatant was prevented by anti-IL-4 mAb (11B11). It was also found that 0.3 to 10 U/ml mouse rIL-4, but none of the rIL-1, IL-2, and IFN-gamma, induced normal T cells to form IgD-BF. Indeed, both IL-2 and IFN-gamma inhibited IL-4 to induce the formation of IgD-BF. In contrast, 10 to 50 U/ml of IFN-gamma induced the formation of IgE-BF, and 50 to 200 U/ml IFN-gamma induced the formation of IgG-BF. However, none of the other lymphokines tested, i.e., IL-1, IL-2, and IL-4, induced the formation of either IgE-BF or IgG-BF. The IgD-BF formed by antigenic stimulation of keyhole limpet hemocyanin-primed spleen cells and those formed by stimulation of normal lymphocytes with 1 to 2 U/ml IL-4 enhanced both IgM and IgG1 plaque-forming cell responses of SRBC-primed spleen cells to homologous Ag. In contrast, 1 to 2 U/ml of IL-4, which could induce the formation of IgD-BF, failed to affect the PFC responses. It appears that the formation of IgD-BF may be involved in the effects of IL-4 to enhance the antibody response.     

Influence of chest background on pulmonary 99mTc-DTPA clearance in interstitial lung disease.

We examined the effect of chest extracellular 99mTc-diethylenetriamine pentaacetate (DTPA) as a background in the measurement of pulmonary 99mTc-DTPA clearance in patients with interstitial lung disease (ILD). Eight healthy nonsmokers (HN) and eight patients with ILD were studied. We monitored changes in gamma counts after the inhalation of 99mTc-DTPA aerosol by using a gamma camera placed over the anterior chest. The rate constant of pulmonary 99mTc-DTPA clearance (k; %/min) was assessed by calculating the slope of the decrease in the gamma counts. The chest background, estimated by 99mTc-DTPA intravenous injection, was subtracted from the original data to obtain the corrected DTPA clearance (kc; %/min). In patients with ILD, k was significantly greater [2.19 +/- 1.03 (SD) %/min; n = 8] compared with HN (0.86 +/- 0.17%/min; n = 8; P < 0.01). In patients with ILD, kc was also greater (2.80 +/- 1.15%/min; n = 8; P < 0.01) compared with HN (1.20 +/- 0.12%/min; n = 8). There was no difference in percent underestimation of k between the two groups (29.1 +/- 8.8% for HN, 22.5 +/- 7.9% for patients with ILD). There was a significant correlation between k and kc among all subjects (r = 0.987, P < 0.01). We conclude that background causes significant underestimation of pulmonary 99mTc-DTPA clearance.     

Immunosuppressive effects of glycosylation inhibiting factor on the IgE and IgG antibody response

Glycosylation inhibiting factor (GIF) was purified from culture filtrates of a T cell hybridoma, 23A4, by affinity chromatography on anti-lipomodulin Sepharose. The factor exhibited phospholipase inhibitory activity upon dephosphorylation. Immunization of BDF1 mice with aluminum hydroxide gel (alum)-absorbed dinitrophenyl derivatives of ovalbumin (DNP-OA) resulted in persistent IgE and IgG antibody formation. However, repeated injections of the affinity-purified GIF into the DNP-OA-primed mice beginning on the day of priming prevented the primary anti-hapten antibody responses of both the IgE and the IgG1 isotypes. Treatment with GIF also diminished on-going IgE antibody formation in the DNP-OA-primed mice. The treatment changed the nature of IgE-binding factors formed by BDF1 spleen cells. Incubation of spleen cells from OA + alum-primed mice with OA resulted in the formation of IgE-potentiating factor, whereas spleen cells of OA-primed, GIF-treated mice formed IgE-suppressive factor upon antigenic stimulation. It was also found that Lyt-2+ T cells in the OA-primed, GIF-treated mouse spleen cells released GIF, which had affinity for OA and bore I-Jb determinant(s). Transfer of a Lyt-1+ cell-depleted fraction of the OA-primed, GIF-treated mouse spleen cells into naive syngeneic animals resulted in suppression of the primary anti-DNP IgE antibody response of the recipients to alum-absorbed DNP-OA, but failed to affect the anti-DNP antibody response to DNP-keyhole limpet hemocyanin. The results indicate that GIF treatment during the primary response to OA facilitated the generation of antigen-specific suppressor T cells.