Human adult tonsil xenotransplantation into SCID mice for studying human immune responses and B cell lymphomagenesis.

OBJECTIVE: To generate a human-mouse xenochimeric model where human cells remain clustered in the animal to optimize their interactions and recovery. MATERIALS AND METHODS: Severe combined immune deficient mice (SCID) were xenografted subcutaneously with human adult tonsil pieces (hu-ton-SCID mice). Such animals were: (a) compared with those receiving tonsil cells in suspension, and (b) immunized with de novo and recall antigens. RESULTS: Human tonsil pieces survived a long period of time in SCID mice, while polyclonal human T- and B-lymphocytes persisted in close vicinity within the implantation area; however, little or no graft-versus-host disease was detectable. Not surprisingly, local development of lymphoproliferative disease was often observed in animals receiving lymphoid implants from donors previously infected by the Epstein-Barr virus. One month after surgery, higher serum levels of human IgG were found in SCID mice transplanted with tonsil pieces (2x10(7) cells/animal) than in animals injected with 5x10(7) tonsil cells in suspension (1.9 vs. 0.3 mg/mL, p < 0.002). Importantly, the production of human IgG in hu-ton-SCID mice remained polyclonal for at least 6 months and was linked to the presence of cells within the implants. Immunization of hu-ton-SCID mice with hepatitis B core, a de novo antigen, did not produce a significant IgG immune response; however immunization with tetanus toxoid (TT), a thymus-dependent recall antigen, yielded high (> 700-fold increase in anti-TT IgG levels) and long-lasting (> 6 months) secondary immune responses. CONCLUSION: The hu-ton-SCID mouse xenochimeric model described in this report may improve our understanding of human lymphoid cell interactions, secondary immune responses, and lymphomagenesis.

Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis.

Hereditary hemochromatosis is a common autosomal recessive disorder of iron metabolism. Recent demonstration of an association between transferrin receptor (TfR) and HFE, a major histocompatibility complex class I-like molecule that has been implicated to play a role in hereditary hemochromatosis, further strengthens the notion that HFE is involved in iron metabolism. Herein we show that TfR is required for and controls the assembly and the intracellular transport and surface expression of HFE. Because surface-expressed HFE and TfR remain firmly associated physically, only the fraction of TfR that is associated with HFE during biosynthesis is affected functionally. Moreover, we show that HFE binding reduces the number of functional transferrin binding sites and impairs TfR internalization, thus reducing the uptake of transferrin-bound iron. Thus, iron homeostasis is indirectly regulated by HFE, a negative modulator of TfR.

Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues.

PAR-2 is a second member of a novel family of G-protein-coupled receptors characterized by a proteolytic cleavage of the amino terminus, thus exposing a tethered peptide ligand that autoactivates the receptor. The physiological and/or pathological role(s) of PAR-2 are still unknown. This study provides tissue-specific cellular localization of PAR-2 in normal human tissues by immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against a peptide corresponding to the amino terminal sequence SLIGKVDGTSHVTGKGV of human PAR-2. Significant PAR-2 immunoreactivity was detected in smooth muscle of vascular and nonvascular origin and stromal cells from a variety of tissues. PAR-2 was also present in endothelial and epithelial cells independent of tissue type. Strong immunolabeling was observed throughout the gastrointestinal tract, indicating a possible function for PAR-2 in this system. In the CNS, PAR-2 was localized to many astrocytes and neurons, suggesting involvement of PAR-2 in neuronal function. A role for PAR-2 in the skin was further supported by its immunolocalization in the epidermis. PAR-2C antibody exemplifies an important tool to address the physiological role(s) of PAR-2.

Lipopolysaccharide binding protein and soluble CD14 receptor protein in amniotic fluid and cord blood in patients at term.

OBJECTIVES: Our purpose was to examine whether lipopolysaccharide binding protein and soluble CD14 are present in amniotic fluid and to determine whether the lipopolysaccharide binding protein and soluble CD14 concentrations are associated with indicators of infection or labor at term. A lipopolysaccharide-lipopolysaccharide binding protein complex activates macrophages through soluble CD14 at lipopolysaccharide concentrations up to 100 times lower than required with lipopolysaccharide alone. Thus lipopolysaccharide binding protein and soluble CD14 in amniotic fluid could explain the high concentrations of cytokines found in amniotic fluid of culture-positive patients and may even explain the presence of cytokines in some culture-negative patients. STUDY DESIGN: Healthy women at term undergoing cesarean section had amniotic fluid, chorioamnion, decidua, and cord blood obtained. Lipopolysaccharide binding protein was measured by enzyme-linked immunosorbent assay. Amniotic fluid was cultured and assayed for cytokines, and the chorioamnion and decidua were cultured and examined histologically. RESULTS: Lipopolysaccharide binding protein and soluble CD14 were present in all amniotic fluids and fetal cord blood. An elevated level of lipopolysaccharide binding protein (270 ng/ml/mg of protein) was present in the amniotic fluid of 12 (36%) of the 33 patients. An elevated level was associated with microorganisms in the chorioamnion and decidua, cytokines (tumor necrosis factor-alpha, interleukin-6, and interleukin-8) in amniotic fluid, histologic chorioamnionitis, and labor. Among patients in labor, the concentration of lipopolysaccharide binding protein appeared independent of microorganisms in the amniotic fluid. CONCLUSIONS: Lipopolysaccharide binding protein and soluble CD14 are present in amniotic fluid, and concentrations of lipopolysaccharide binding protein are elevated in patients in labor with and without evidence of infection. Lipopolysaccharide binding protein and soluble CD14 may mediate intrauterine inflammatory responses at term.

Bronchoalveolar macrophage CD14 expression: shift between membrane-associated and soluble pools.

The bacterial endotoxin [lipopolysaccharide (LPS)]-binding protein CD14 modulates the host response to LPS, but membrane-associated and soluble forms of the molecule exert different biological effects. CD14 anchored to the mononuclear phagocyte membrane (mCD14) enhances response to LPS. Soluble CD14 (sCD14) may block LPS stimulation of CD14-bearing cells while supporting LPS presentation to non-CD14-bearing cells. We analyzed cell mCD14 and sCD14 expression in simultaneously collected human bronchoalveolar macrophages (BAM) and peripheral blood monocytes (PBM). Expression of mCD14 in freshly isolated BAM was only 9% as high as in PBM. Levels of sCD14 in 48 h in BAM culture supernatants were 19% as high as in PBM cultures. Interleukin (IL)-6 increased CD14 expression in both BAM and PBM but exerted different effects on CD14 distribution in these cell types. IL-6 increased only sCD14 release (2.5-fold) in BAM while increasing only mCD14 expression (2.5-fold) in PBM. IL-4 reduced both mCD14 (> 40%) and sCD14 (> 60%) expression in both cell types. We speculate that the balance between sCD14 and mCD14 expression influences the response to aspirated or inhaled LPS in the bronchoalveolar compartment. Cytokine expression and monocyte recruitment may influence this process by modulating CD14 expression.

Relationship between soluble CD14, lipopolysaccharide binding protein, and the alveolar inflammatory response in patients with acute respiratory distress syndrome.

The effects of bacterial endotoxin (lipopolysaccharide, LPS) are amplified by lipopolysaccharide binding protein (LBP) and CD14, resulting in cellular activation at very low concentrations of LPS. To investigate the importance of this pathway in acute lung injury, we measured LPS, LBP, and soluble CD14 (sCD14) in the bronchoalveolar lavage fluid (BAL) of 82 patients with acute respiratory distress syndrome (ARDS). LBP and sCD14 increased markedly in BAL of patients with ARDS. sCD14 and LBP each were strongly related to BAL total protein and polymorphonuclear neutrophil (PMN) concentration, whereas LPS concentration was not. Multivariate analyses showed sCD14 to be strongly related to BAL total protein, even after controlling for LPS and LBP concentrations. sCD14 was strongly and independently related to PMN concentration, after controlling for BAL LPS, LBP, and interleukin-8 (IL-8). The BAL LPS concentration was not strongly related to either BAL total protein or BAL PMN. The BAL sCD14 and LBP values were similar in all subgroups of patients with ARDS, and were not related to survival. The serum LBP and sCD14 were elevated in ARDS, but were not related to BAL total protein, LBP, sCD14, PMN, or clinical outcome. Thus, LBP and sCD14 reach high concentrations in the lungs of patients with ARDS, and BAL sCD14 is strongly related to two major indices of lung inflammation: total protein and PMN concentration. CD14-dependent mechanisms may contribute to lung inflammation in ARDS.

Antibodies against CD14 protect primates from endotoxin-induced shock.

Lipopolysaccharide (LPS), residing in the outer membrane of all gram-negative bacteria, is considered a major initiating factor of the gram-negative septic shock syndrome in humans. LPS forms a complex with the LPS binding protein (LBP) in plasma, and LPS-LBP complexes engage a specific receptor, CD14, on the surface of myeloid cells, leading to the production of potent proinflammatory cytokines. The major goal of this study was to test the importance of the CD14 pathway in vivo in a primate model that is similar to human septic shock. Primates were pretreated with one of two different inhibitory anti-CD14 mAbs, then challenged with intravenous endotoxin (375 microg/kg/h) for 8 h. The anti-CD14 treatment regimens were successful in preventing profound hypotension, reducing plasma cytokine levels (TNF-alpha, IL-1beta, IL-6, and IL-8), and inhibiting the alteration in lung epithelial permeability that occurred in animals treated with LPS and an isotype-matched control antibody. These results demonstrate for the first time the importance of the CD14 pathway in a primate model that is similar to human septic shock. Inhibition of the CD14 pathway represents a novel therapeutic approach to treating this life-threatening condition.

In vivo characterization of the proteasome regulator PA28.

A proteasome regulator, termed PA28, has been shown to modulate peptidase activities of the proteasomes in vitro. Two different but homologous PA28 molecules, designated as PA28alpha and PA28beta, have been cloned. Both alpha and beta polypeptides of PA28 are found in PA28 complexes isolated from cells, indicating that both are constituents of functional PA28 complexes. Using antisera specific to PA28alpha, PA28beta, and epitope-tagged PA28 molecules, we show that expression of PA28alpha and PA28beta is coordinately induced by various cytokines in different cell lines and that PA28 subunits and proteasomes have almost identical half-lives. In addition, we show that PA28 complexes are associated with 20 S but not 26 S proteasomes in vivo. Moreover, we demonstrate that PA28 complex is a heterohexamer composed of both alpha and beta subunits with a stoichiometry of alpha3beta3 in an alternating order.

Asthma and endotoxin: lipopolysaccharide-binding protein and soluble CD14 in bronchoalveolar compartment.

In allergic asthma, inhalation of antigen provokes an early increase in microvascular permeability with protein extravasation and a delayed recruitment of inflammatory cells. We showed that similar concentrations of lipopolysaccharide (LPS) are present in bronchoalveolar lavage fluid (BALF) in 12 subjects without asthma (86.5 +/- 53.8 pg/ml) and 12 subjects with mild asthma (111 +/- 37.0 pg/ml). These LPS levels are insufficient to stimulate cytokine release without accessory molecules. BALF obtained 24 h after segmental ragweed antigen challenge in 11 asthmatics allergic to ragweed contained increased levels of two LPS accessory molecules compared with preantigen BALF, 158-fold more LPS-binding protein (LBP) 4.83 +/- 2.02 vs. 742 +/- 387 ng/ml; P < 0.03) and 31.6-fold more soluble CD14 (sCD14) (3.45 +/- 1.04 vs. 110 +/- 51.6 ng/ml; P < 0.02). Postantigen BALF enhanced binding of fluorescein-conjugated LPS to CD14-bearing THP-1 cells and supported LPS-induced non-CD14-bearing endothelial cell expression of intercellular adhesion molecule-1 and interleukin-6, indicating functional LBP and sCD14. We suggest that extravasation of LBP and sCD14 into the bronchoalveolar compartment after antigen inhalation may enhance the capacity of inhaled or aspirated LPS to activate an inflammatory cascade that may amplify the inflammatory response to inhaled antigen in some asthmatics.

Antigen presentation and T cell development in H2-M-deficient mice.

HLA-DM (DM) facilitates peptide loading of major histocompatibility complex class II molecules in human cell lines. Mice lacking functional H2-M, the mouse equivalent of DM, have normal amounts of class II molecules at the cell surface, but most of these are associated with invariant chain-derived CLIP peptides. These mice contain large numbers of CD4+ T cells, which is indicative of positive selection in the thymus. Their CD4+ cells were unresponsive to self H2-M-deficient antigen-presenting cells (APCs) but were hyperreactive to wild-type APCs. H2-M-deficient APCs failed to elicit proliferative responses from wild-type T cells.

The CD14 differentiation antigen mediates the development of endotoxin responsiveness during differentiation of mononuclear phagocytes.

The CD14 antigen was originally described as a differentiation antigen on mononuclear cells. The purpose of this study was to investigate the relationship between the appearance of surface CD14 and the acquisition of lipopolysaccharide (LPS) responsiveness during maturation of mononuclear phagocytes. Immature THP-1 cells responded poorly to LPS in the absence or presence of serum. Treatment with the maturational agent calcitriol caused a dose- and time-dependent increase in CD14 mRNA and surface CD14 and enhanced the responsiveness of THP-1 cells to smooth and rough form LPS, complexes of LPS and lipopolysaccharide-binding protein (LBP), and LPS in low concentrations of serum. Monoclonal antibodies to CD14 blocked the responses of THP-1 to LPS, LPS-LBP complexes and LPS in serum. Immunodepletion of LBP from serum also inhibited the effect of LPS in serum. The data show that maturation of the response of THP-1 cells to LPS and LPS-LBP complexes depends on the appearance of CD14 on the cell surface. Maturation of the response to LPS in serum depends in large part on the appearance of CD14 on the cell surface and the presence of LBP in serum.

Analysis of lipopolysaccharide binding by CD14.

The cell surface protein CD14 binds bacterial lipopolysaccharide (LPS) in the presence of the serum protein, LPS-binding protein (LBP). This interaction is important for LPS-induced activation of mammalian myeloid cells. We performed quantitative studies of 3H-labeled LPS binding to human CD14 expressed on Chinese hamster ovary cells and on a human macrophage cell line (THP-1). At the concentrations studied (20-100 nM) LPS binding required the expression of CD14 and could be inhibited by a subset of anti-CD14 monoclonal antibodies. LBP was required for LPS binding to CD14. The binding occurred within 10 min and was relatively unaffected by temperature over the range of 4-37 degrees C. Quantitative binding assays were performed at 10 degrees C, or at 37 degrees C, using Chinese hamster ovary cells depleted of ATP. In both cases, 75-90% of the LPS could be released by treatment with phosphatidylinositol-specific phospholipase C, suggesting that it remains associated with the glycosyl phosphatidylinositol-anchored CD14. The apparent dissociation constant of recombinant human CD14 expressed on Chinese hamster ovary cells for LPS at 10 degrees C was 2.74 (+/- 0.99) x 10(-8) M; the apparent dissociation constant of CD14 expressed on THP-1 cells at 10 degrees C was 4.89 (+/- 1.42) x 10(-8) M. In both cell lines, at saturating LPS concentrations, the molar ratio of LPS bound per surface CD14 was approximately 20:1. At 37 degrees C the apparent dissociation constant of recombinant human CD14 for LPS at 37 degrees C was 2.7 (+/- 1.2) x 10(-8) M, and the molar ratio of LPS bound per surface CD14 was approximately 8:1. Although the difference in molar ratio of LPS bound per surface CD14 at the two temperatures is difficult to interpret, it is clear that at both temperatures the molar ratio is not 1:1. The basis of this phenomenon is unclear, but may involve the repeated leucine-rich motifs, which are found within CD14.

Glycosyl-phosphatidylinositol-anchored or integral membrane forms of CD14 mediate identical cellular responses to endotoxin.

Endotoxin stimulates leukocytes to release cytokines that initiate septic shock in humans and animals. CD14, a glycosyl-phosphatidylinositol-anchored membrane glycoprotein, is an endotoxin receptor on leukocytes, and endotoxin binding to CD14 induces cytokine production. Here we show that glycosyl-phosphatidylinositol-anchored or integral membrane CD14 mediates identical cellular responses to endotoxin, including NF-kappa B activation and protein tyrosine phosphorylation. We also show that an anti-CD14 monoclonal antibody that does not block endotoxin binding to CD14 nonetheless inhibits cell activation by endotoxin. These findings suggest that binding of endotoxin to cell-surface CD14 is followed by subsequent interactions of the endotoxin-CD14 complex with additional membrane component(s) that enable transmembrane signaling. This function of CD14 may be prototypic for other members of the glycosyl-phosphatidylinositol-anchored family of proteins that do not play a primary role in signal transduction but rather are the principal ligand-binding units of membrane-bound receptor complexes.

Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14.

Myeloid cell activation by lipopolysaccharides (LPS) involves two proteins, plasma LPS-binding protein (LBP) and cell-membrane CD14. Cell membrane CD14, anchored by a glycerophosphatidylinositol tail, is the cellular receptor for LPS-LBP complexes. Another form of CD14, without the lipid tail, circulates as a soluble plasma protein. In this work we show that soluble CD14 (sCD14) is required for activation of endothelial and epithelial cells by LPS. We propose that LPS-LBP complexes transfer LPS to sCD14, and the LPS-sCD14 complexes then bind to a cellular receptor. Support for this pathway comes from experiments in which LBP and CD14 in normal human serum are blocked by specific antibodies, experiments in which serum is replaced by purified LBP and sCD14, and experiments in which specific binding of [3H]LPS to epithelial cells is quantitated.

Lipopolysaccharide binding protein enhances the responsiveness of alveolar macrophages to bacterial lipopolysaccharide. Implications for cytokine production in normal and injured lungs.

A plasma lipopolysaccharide (LPS)-binding protein (LBP) has been shown to regulate the response of rabbit peritoneal macrophages and human blood monocytes to endotoxin (LPS). We investigated whether LBP is present in lung fluids and the effects of LBP on the response of lung macrophages to LPS. Immunoreactive LBP was detectable in the lavage fluids of patients with the adult respiratory distress syndrome by immunoprecipitation followed by Western blotting, and also by specific immunoassay. In rabbits, the LBP appeared to originate outside of the lungs, inasmuch as mRNA transcripts for LBP were identified in total cellular RNA from liver, but not from lung homogenates or alveolar macrophages. Purified LBP enhanced the response of human and rabbit alveolar macrophages to both smooth form LPS (Escherichia coli O111B:4) and rough form LPS (Salmonella minnesota Re595). In the presence of LBP and LPS, the onset of tumor necrosis factor-alpha (TNF alpha) production occurred earlier and at an LPS threshold dose that was as much as 1,000-fold lower for both types of LPS. In rabbit alveolar macrophages treated with LBP and LPS, TNF alpha mRNA appeared earlier, reached higher levels, and had a prolonged half-life as compared with LPS treatment alone. Neither LPS nor LPS and LBP affected pHi or [Cai++] in alveolar macrophages. Specific monoclonal antibodies to CD14, a receptor that binds LPS/LBP complexes, inhibited TNF alpha production by human alveolar macrophages stimulated with LPS alone or with LPS/LBP complexes, indicating the importance of CD14 in mediating the effects of LPS on alveolar macrophages. Thus, immunoreactive LBP accumulates in lung lavage fluids in patients with lung injury and enhances LPS-stimulated TNF alpha gene expression in alveolar macrophages by a pathway that depends on the CD14 receptor. LBP may play an important role in augmenting TNF alpha expression by alveolar macrophages within the lungs.

The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.