Contribution of FcRn binding to intestinal uptake of IgG in suckling rat pups and human FcRn-transgenic mice.

Immunoglobulin G (IgG) is transcytosed across intestinal epithelial cells of suckling mammals by the neonatal Fc receptor (FcRn); however, the contribution of FcRn vs. FcRn-independent uptake to serum IgG levels had not been determined in either rat pups or human (h)FcRn-expressing mice (Tg276 and Tg32). In isoflurane-anesthetized rodents, serum levels were determined after regional intestinal delivery of human monoclonal antibodies (hIgG) with either wild-type (WT) Fc sequences or variants engineered for different FcRn binding affinities. Detection of full-length hIgG was by immunoassay; intestinal hFcRn and hIgG localization was by immunocytochemistry. High (µg/ml) serum levels of hIgG were detected after proximal intestinal delivery (0.1-10 mg/kg) in 2-wk-old rats. Human FcRn was visualized in epithelial cells of Tg276 mice, but low serum hIgG levels (<10 ng/ml) were obtained. In rat pups, intraintestinal hIgG1 WT administration resulted in dose-related and saturable uptake, whereas uptake of a low FcRn-binding affinity variant was nonsaturable. There were no differences in hIgG levels from systemic and hepatic portal vein serum samples, and intense hIgG immunostaining was noted in villi enterocytes and within lymphatic lacteal-like vessels. This study demonstrated that FcRn-mediated uptake in rat pups accounted for ~80% of serum hIgG levels and that IgG enters the circulation via the lymph and not the hepatic portal vein. The remaining uptake though the immature intestine is nonreceptor mediated. Intestinal epithelial cell hFcRn expression occurred in Tg276 mice, but receptor-mediated transport of IgG was not observed. The suckling rat pup intestine is a mechanistic model of FcRn-IgG-mediated transcytosis.

IkappaBalpha deficiency results in a sustained NF-kappaB response and severe widespread dermatitis in mice.

The ubiquitous transcription factor NF-kappaB is an essential component in signal transduction pathways, in inflammation, and in the immune response. NF-kappaB is maintained in an inactive state in the cytoplasm by protein-protein interaction with IkappaBalpha. Upon stimulation, rapid degradation of IkappaBalpha allows nuclear translocation of NF-kappaB. To study the importance of IkappaBalpha in signal transduction, IkappaBalpha-deficient mice were derived by gene targeting. Cultured fibroblasts derived from IkappaBalpha-deficient embryos exhibit levels of NF-kappaB1, NF-kappaB2, RelA, c-Rel, and IkappaBbeta similar to those of wild-type fibroblasts. A failure to increase nuclear levels of NF-kappaB indicates that cytoplasmic retention of NF-kappaB may be compensated for by other IkappaB proteins. Treatment of wild-type cells with tumor necrosis factor alpha (TNF-alpha) resulted in rapid, transient nuclear localization of NF-kappaB. IkappaBalpha-deficient fibroblasts are also TNF-alpha responsive, but nuclear localization of NF-kappaB is prolonged, thus demonstrating that a major irreplaceable function Of IkappaBalpha is termination of the NF-kappaB response. Consistent with these observations, and with IkappaBalpha and NF-kappaB's role in regulating inflammatory and immune responses, is the normal development Of IkappaBalpha-deficient mice. However, growth ceases 3 days after birth and death usually occurs at 7 to 10 days of age. An increased percentage of monocytes/macrophages was detected in spleen cells taken from 5-, 7-, and 9-day-old pups. Death is accompanied by severe widespread dermatitis and increased levels of TNF-alpha mRNA in the skin.

Heterogeneity among T helper cells. Interleukin 2 secretion and help for immunoglobulin secretion.

T lymphocytes are thought to provide "help" for B cells by activating them from the resting state, by secretion of antigen-nonspecific lymphokines that promote B cell differentiation and maturation, and by providing signals that induce isotype switching. To clarify the extent to which these different forms of helper activity could be carried out by individual T cells, we set up cultures in which B cells activated, and were in turn themselves stimulated by, limiting numbers of T cells through differences at the H-2 or Mls loci. At T cell doses at which responses were likely to represent the activity of individual helper T cells (or their immediate clonal progeny), we found that some T cells were able both to produce interleukin 2 (IL-2) and to induce secretion of both IgM and IgG, whereas others induced immunoglobulin (Ig) secretion without detectable IL-2 production, and still others made IL-2 but did not promote antibody secretion. We could not detect B cell stimulatory factor 1 production by alloantigen-stimulated T cells, and the addition of antibodies to B cell stimulatory factor 1 did not prevent Ig production. Two results, however--higher Ig accumulation in those wells that received an IL-2-producing cell, and inhibition by anti-IL-2 receptor antibodies of B cell but not T cell function--are consistent with a direct stimulatory effect of IL-2 on B cells in this system. The pattern of helper functions exhibited by T cells freshly isolated from mice differs from that inferred from studies of cloned lines of T cells in long term cultures.

Frequencies of IL-2- and IL-4-secreting T cells in naive and antigen-stimulated lymphocyte populations.

The relative frequencies of IL-2-and IL-4-secreting precursors in naive and Ag-primed populations were investigated by using limiting dilution analysis. Cells capable of IL-4 production in lymphoid populations freshly isolated from mice were rare in comparison with those producing IL-2 when the cells were stimulated by nominal Ag, by alloantigens, or by mitogens. One cycle of in vitro Ag restimulation and rest, however, enabled us to detect high proportions of IL-4-secreting cells among keyhole limpet hemocyanin-primed lymph node cells. With cultures set up at monoclonal cell doses, it was shown that IL-2 and IL-4 are secreted by separate precursor populations at this stage of their development. The IL-4-secreting cells were further shown to be dependent upon the presence of IL-2, either secreted by separate precursors or exogenously added, for the production of detectable amounts of IL-4. Analysis of the frequencies of helper cells producing both IL-2 and IL-4 at various stages of the in vivo immune response and the requirements for their growth and differentiation should give a better understanding of the relative contributions of each cell type.

Interleukin-1 (IL-1) receptors on murine splenic B cells and the 70Z/3 pre-B-cell line appear to be identical and bind human IL-1 alpha and IL-1 beta differentially.

In this study murine splenocytes were found to possess specific, high-affinity IL-1 receptors (IL-1R) capable of binding radiolabeled human IL-1 alpha with a Kd of 2-6 x 10(-10) M. Experiments performed with purified splenic B cells demonstrated that B cells express low levels of IL-1R (100-200 receptors per cell). Separation of splenic B cells into high- (resting) and low-density (in vivo-activated) fractions showed that low-density B cells expressed 2-fold more IL-1R compared with high-density B cells suggesting that IL-1R are upregulated on B-cell activation. In vitro stimulation of B cells with mitogens resulted in a 5- to 10-fold increase in IL-1R expression compared to IL-1R levels on unstimulated B cells. Receptors on the murine pre-B-cell line 70Z/3, which possesses type II IL-1R, and murine splenic B cells appear to be identical. Cross-linking studies demonstrated that the 125I-labeled IL-1/IL-1R complex on B cells and 70Z/3 IL-1R was found to block binding of IL-1 to IL-1R on 70Z/3 and splenic B cells, but not to type I IL-1R on murine EL4 thymoma cells. Competitive inhibition experiments showed differential binding of human IL-1 beta to splenic B cells and 70Z/3 cells as a function of temperature. The IL-1R on 70Z/3 and splenic B cells bound human IL-1 alpha, murine IL-1 alpha, and murine IL-1 beta with high affinity at both 4 degrees and 37 degrees C. In contrast, the affinity of IL-1R on these same cell types for human IL-1 beta was significantly reduced at 37 degrees C compared to 4 degrees C. The reduced binding affinity for human IL-1 beta was due to an increased off-rate at 37 degrees C compared with 4 degrees C. Characterization of IL-1R on murine splenic B cells will help clarify the role of IL-1 in the regulation of the immune response.

Expression of soluble human interleukin-2 receptor alpha-chain in Escherichia coli.

A soluble, biologically active form of IL-2R alpha known as delta MST and consisting of the 178 N-terminal amino acid residues of the mature protein was directly expressed in the cytoplasm and the periplasm of Escherichia coli. Because it was not glycosylated, the E. coli protein was substantially less heterogeneous than delta MST expressed in insect cells. Nevertheless, it manifested equivalent biological activity in an IL-2 binding assay. The level of active delta MST production was higher when the protein was expressed in secretable form with a bacterial signal peptide than when it was produced in the cytoplasm, probably because the oxidizing environment and the presence of disulfide isomerases in the periplasm facilitated the correct folding of delta MST.

Monoclonal antibody 2D10 recognizes a novel T cell costimulatory molecule on activated murine B lymphocytes.

We have developed a panel of rat mAbs against dibutyryl cAMP-activated 5C2 cells. In this panel, one mAb, 1G10, recognized murine B7. Another mAb designated 2D10 did not bind to murine B7 but could recognize a surface molecule expressed only on dibutyryl cAMP-activated 5C2 mouse B lymphoma cells or on LPS-stimulated splenic B cells. This new molecule is referred to as early T cell costimulatory molecule-1 (ETC-1). From both activated 5C2 cells and splenic B cells, mAb 2D10 immunoprecipitated a 59- to 60-kDa protein, which was different from the 47- to 55-kDa murine B7 protein precipitated from the same cell populations. FACS analysis showed that, in contrast to B7, the expression of ETC-1 on 5C2 cells was induced by lower concentrations of dibutyryl cAMP and displayed a faster kinetics. LPS-stimulated splenic B cells expressed relatively low levels of B7 and much higher levels of ETC-1. Importantly, in an Ag presentation assay using activated 5C2 cells as APC, the secretion of IL-2 by C8A3 T hybrids was partially inhibited by mAb 2D10 alone and completely blocked by combination use of mAbs 2D10 and 1G10 in a dose-dependent and synergistic fashion. In a one-way primary MLR, mAb 2D10 alone at 0.1 to 1 microgram/ml inhibited T cell proliferation by 19 to 56%. However, an additive blocking effect (up to 76%) was observed when two mAbs were added in combination. Thus, our data have demonstrated that a novel T cell costimulatory molecule is present on activated murine B cells, which, in cooperation with B7, may play a critical role in optimal T cell activation.

Expression and functional analysis of murine B7 delineated by a novel monoclonal antibody.

The B cell surface molecule designated B7 has been shown to be expressed by activated human B cells and monocytes and to be a ligand for the CD28 and CTLA-4 molecules on T cells. B7/CD28 interactions can provide a second signal to T cells (in addition to occupancy of the T cell antigen receptor) that is needed for T cell activation. COS cells transfected with the mouse homologue of B7 have been demonstrated to provide a stimulatory signal to murine and human T cells. In this report we describe a rat anti-mouse B7 mAb designated 1G10. Scatchard and/or FACS analyses utilizing 1G10 demonstrated that B7 was not expressed on resting splenic T cells or B cells, but could be induced at high levels on B cells cocultured with a syngeneic I-Ak-restricted autoreactive T cell hybridoma. Furthermore, activation of B cells with dibutyryl-cAMP (db-cAMP), a second messenger for class II MHC signaling, or with LPS induced the expression of B7 and the two agents showed additive effects. In contrast to B cells, freshly isolated mouse peritoneal macrophages constitutively expressed B7. Antibody-blocking experiments indicated that anti-B7 antibody partially inhibited T cell proliferative responses to primary antigenic stimulation but had no effect on the responses of previously activated T cells to antigenic restimulation.