Interleukin-12 acts as an adjuvant for humoral immunity through interferon-gamma-dependent and -independent mechanisms.

Interleukin-12 (IL-12) is a pivotal cytokine that has dramatic effects on cell-mediated immunity. It is now becoming increasingly recognized that IL-12 also strongly controls humoral immunity. We have investigated the mechanism by which IL-12 induces alterations in antibody isotype expression by determining the influence of IL-12 on in vitro immunoglobulin (Ig) production in polyclonally activated murine spleen cell cultures. Cells exposed to IL-12 plus lipopolysaccharide or anti-CD40 monoclonal antibody showed dramatically elevated IgG2a and suppressed IgG1 production compared to cells cultured in the absence of IL-12. IL-12 treatment of spleen cell cultures induced expression of gamma2a germ-line transcripts, consistent with initiation of switch recombination to IgG2a. In addition, exposure of limiting dilution cultures to IL-12 increased IgG2a+ cell precursor frequency. All of the above results were dependent on interferon-gamma (IFN-gamma). However, in the absence of IFN-gamma, IL-12 still had significant effects on Ig secretion. Specifically, IL-12 enhanced IgG1 and IgG2b anti-DNP antibody levels in mice containing specific disruptions in the IFN-gamma gene. Our results suggest that IL-12 induces T helper type 1 and natural killer cells to secrete large amounts of IFN-gamma which then causes B cells to switch to IgG2a and IgG3 production. In addition, IL-12 has direct or indirect effects on B cells that are independent of IFN-gamma. The IFN-gamma-independent effects may include enhancement of Ig expression by post-switched cells.

Human APOE protein localized in brains of transgenic mice.

Transgenic mice carrying the three common human apolipoprotein E (APOE) alleles have been developed. In this study, brains of the transgenic mice have been analyzed by in situ histohybridization, immunohistochemistry, and immunoblots to determine sites of gene expression, to identify specific brain cells associated with human apoE protein, and to determine the relative concentrations of the human apoE. Results indicate that (1) human APOE mRNA and apoE protein occur in the gray and white matter of transgenic mouse brains; (2) in the hippocampus of transgenic brains, human apoE protein reacts immunologically within the same cells as the glial fibrillary acidic protein (GFAP), a specific marker for astrocytes; and (3) concentrations of the apoE isoforms determined in three heterozygous transgenic brains range from 22 to 250 pmol/g wet weight of brain.

Enhancement of humoral immunity by interleukin-12.

We have found that IL-12 treatment of mice leads to long-lasting enhancement in production of most antibody isotypes in conventional B-cell responses. Initial recruitment of new B-cell clones into the response is mediated by IFN-gamma, but subsequent enhancement of Ig secretion appears to be IFN-gamma-independent. We have further found that activated B cells can directly bind IL-12. Taken together, our results suggest a two-step model for the role of IL-12 in enhancement of humoral immunity. Initially, IL-12 induces production of IFN-gamma from Th1 and NK cells. Enough cytokine can be produced from either cell type to then mediate gamma 2a heavy chain isotype switching as well as temporary suppression of IgG1 production. IL-12 further stimulates post-switched cells, including cells producing IgG1, to secrete greatly increased amounts of antibody. This step is not mediated by IFN-gamma but might be due to direct IL-12 binding to activated B lymphocytes. Depletion of B1 cells by IL-12 may further enhance antibody responsiveness since B1 cells are known to competitively inhibit Ig secretion by conventional B cells. The end result is that IL-12 causes a generalized upregulation in production of all antibodies and therefore acts as a strong adjuvant for humoral as well as cellular immunity.

Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles.

Transgenic mice carrying heterologous genes directed by a 670-bp segment of the regulatory sequence from the human transferrin (TF) gene demonstrated high expression in brain. Mice carrying the chimeric 0.67kbTF-CAT gene expressed TF-CAT in neurons and glial cells of the nucleus basalis, the cerebrum, corpus callosum, cerebellum, and hippocampus. In brains from two independent TF-CAT transgenic founder lines, copy number of TF-CAT mRNA exceeded the number of mRNA transcripts encoding either mouse endogenous transferrin or mouse endogenous amyloid precursor protein. In two transgenic founder lines, the chloramphenicol acetyltransferase (CAT) protein synthesized from the TF-CAT mRNA was estimated to be 0.10-0.15% of the total soluble proteins of the brain. High expression observed in brain indicates that the 0.67kbTF promoter is a promising director of brain expression of heterologous genes. Therefore, the promoter has been used to express the three common human apolipoprotein E (apoE) alleles in transgenic mouse brains. The apoE alleles have been implicated in the expression of Alzheimer disease, and the human apoE isoforms are reported to interact with different affinities to the brain beta-amyloid and tau protein in vitro. Results of this study demonstrate high expression and production of human apoE proteins in transgenic mouse brains. The model may be used to characterize the interaction of human apoE isoforms with other brain proteins and provide information helpful in designing therapeutic strategies for Alzheimer disease.

Interleukin 12 alters the isotype-restricted antibody response of mice to hen eggwhite lysozyme.

Protein antigens elicit humoral responses in mice that consist predominantly of IgG1 antibodies. We have now investigated the ability of IL-12, a cytokine reported to augment IgG2a anti-hapten responses through activation of Th1 cells, to alter antibody responses to hen eggwhite lysozyme (HEL). The normal response of BALB/c mice to HEL is highly restricted to IgG1 expression and therefore provides an excellent system for determining effects of cytokines on expression of other isotypes. Seven days after immunization, IL-12 treated mice demonstrated greatly elevated HEL-specific IgG2a antibody levels and suppressed IgG1 production, while PBS-treated control mice showed a typical IgG1-restricted response. On day 28, IL-12-treated mice showed heightened serum antibody levels of both isotypes. Delaying cytokine treatment until after the typical IgG1 anti-HEL response had already been established also led to significant elevation of serum IgG2a antibody levels. These effects correlated with increased IFN-gamma production; however, administration of IL-12 plus anti-IFN-gamma had little influence on IgG2a enhancement, although it did relieve the early IgG1 suppression. Furthermore, the differential effects of Il-12 on isotype expression did not correlate with time; in fact, IgG2a enhancement correlated with loss of IgG1 suppression. Our findings indicate that (i) IL-12 reproducibly induces large amounts of IgG2a HEL-specific antibodies in vivo; (ii) it can alter isotype profiles of both primary and secondary responses; and (iii) its effects on humoral immunity are not completely explained by induction of Th1 cell derived IFN-gamma.

A human (3.3 kb) haptoglobin-CAT transgene is modulated in lungs of transgenic mice by inflammation.

Four independent lines of transgenic mice were produced carrying integrated copies of a chimeric gene composed of 3.3 kb of the human haptoglobin 5' regulatory region fused to the CAT (chloramphenicol acetyl transferase) reporter gene. Although the endogenous mouse haptoglobin (Hp) and human haptoglobin (HP) genes express mainly in liver and lung, expression of the human 3.3-kb HP-CAT transgene was not detected until after induction of inflammation and then only in lungs. The results indicated that the transgene maintained the regulatory DNA elements required for lung specific responsiveness to inflammation in vivo but lacked the DNA sequence required for robust expression in liver. The DNA sequence(s) responsible for the normally high level of HP expression in liver either reside outside the 3.3-kb regulatory region of the HP chimeric gene or this region contains a suppressor sequence affecting tissue specific expression in the liver.

IL-1 beta decreases expression of amyloid precursor protein gene in human glioma cells.

In Alzheimer's disease a small fragment of the amyloid protein precursor (APP), called beta 4, is a characteristic component of senile plaques in brains of affected patients. Efforts to intervene in Alzheimer's disease include approaches by which APP levels can be decreased in brain. The study described here demonstrates the expression of APP gene in four cell lines that originated from human brain glioblastomas. In one line, HTB 17, APP mRNA level was approximately 25% the APP mRNA found in human brain and 150% that found in human liver. To ascertain whether or not APP expression in HTB 17 cells could be modulated by a cytokine associated with the inflammatory response, cells were cultured in the presence of IL-1 beta. A significant decrease in APP mRNA accompanied treatment of glioma cells with IL-1 beta.

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.

Tissue specific expression of mouse transferrin during development and aging.

Transferrin (TF) is a major plasma protein that binds ferric iron and transports it to all target tissues of the body. This study is the first step to identify the tissue specific expression of the transferrin gene in mice during development, into maturity and throughout the aging process. The transferrin gene expresses mainly in mouse liver, the cerebral hemispheres and cerebellum. In mouse, transferrin is expressed in peritoneal macrophages and in mouse macrophage cell line MO59. At 19 days of gestation, transferrin mRNA is detected in the fetal lung, heart, stomach and kidney. TF mRNA levels increase in liver throughout gestation with maximum expression occurring at 19 days. Transferrin mRNA was detected in placentas of pregnant mice, with levels progressively increasing throughout the term of pregnancy. The levels of liver TF mRNA in mouse vary in a cyclic manner during the development increasing with the aging processes. Because of the dynamic nature of tissue requirements for transferrin during homeostasis the TF gene serves as a promising system for analyzing tissue-specific regulation in vivo during development and aging. Results from this study designate periods in the life-span of the mouse where regulatory mechanisms interacting with the TF gene appear to dynamically alter its expression.

Expression of transferrin and vitamin D-binding protein genes in an osteogenic sarcoma cell line.

Expression of genes encoding transferrin and the vitamin D-binding protein is described in a cell line, U-2 OS, derived from a human osteogenic sarcoma. The mRNA transcripts of transferrin and vitamin D-binding protein were shown to be the lengths of those found in normal human liver. The cells synthesize and secrete the transferrin and vitamin D-binding proteins, in addition to human albumin and ceruloplasmin. The U-2 OS cells were successfully transfected with chimeric genes carrying 670 bp of the 5' regulatory sequence of the human transferrin gene fused to a reporter chloramphenicol acetyltransferase gene. These data indicate that the appropriate transcriptional factors required for expression of four plasma proteins are produced by U-2 OS nuclei and that the U-2 OS cell line will be useful for studies analyzing regulation of these genes.

Human interleukin 3: effects on normal and leukemic cells.

The effects of recombinant human interleukin 3 (IL3) on normal bone marrow cells and human leukemic cells were studied. In clonal assays, IL3 supported the growth of all colony types including megakaryocytes. Erythroid colonies were formed in the presence of IL3 and erythropoietin, but not in the absence of erythropoietin. Replating experiments using blast cell colonies derived from a cell population enriched for progenitor cells by fluorescence-activated cell sorting with the monoclonal antibody 3C5, showed that IL3 supported the continued replating of colonies. The clonal proliferation of human bone marrow cells in response to IL3 was inhibited by tumor necrosis factor and by lymphotoxin, but not by interferon-gamma. In suspension cultures, IL3 supported the proliferation of mast cells. Human IL3 had no effect on the growth responses, morphology, cytochemistry, or clonogenicity of the human leukemic cell lines HL60, U-937, KG1a, and HEL. Transcripts for IL3 mRNA were not detectable in these cells, nor in the K562 cell line, implying that autocrine secretion of IL3 was not the mechanism by which these leukemias were maintained. Although cells derived from the bone marrow or peripheral blood of twenty patients with myeloproliferative disorders, myelodysplastic syndromes or acute myeloid leukemia frequently showed proliferative responses to IL3, mRNA transcripts for IL3 were not detected in these cells.

Postoperative pain relief; a new approach: narcotics compared with non-steroidal anti-inflammatory drugs.

The pain relief provided by regular intramuscular diclofenac and on demand intramuscular papaveretum was compared over a 48 h postoperative period in 114 patients undergoing total hip replacement. The study was of a randomised, single-blind, between-group design. Patients were assessed by a surgeon, physiotherapist and nursing staff. Diclofenac was more effective than papaveretum in pain control (P less than 0.001), wound tenderness (P less than 0.01), awareness (P less than 0.001) and mobilisation (P less than 0.01). Wound drainage (P greater than 0.05) and wound oedema (P greater than 0.05) were not significantly different in the two treatments. Gastrointestinal complications were encountered in both groups; two patients on diclofenac had to be withdrawn because of them. The use of diclofenac given as a postoperative analgesic is rewarding, particularly in patients undergoing musculoskeletal procedures. Patients will be more comfortable and will mobilise better during their whole postoperative course.

A controlled prospective study of early mobilization of minimally displaced fractures of the distal radial metaphysis.

A controlled prospective trial of early exercise of slightly displaced fractures of the distal radial metaphysis has been performed. Twenty-eight fractures were enclosed in a tubigrip support 7-13 days after injury and 28 fractures were splinted in below-elbow plaster for 4 weeks. It was found that early movement of the wrist produced a superior functional result at 5 and 7 weeks (P less than 0.05) and allowed an earlier recovery of domestic skills (P less than 0.05). Early exercise did not improve recovery of strength of grip and did not produce a higher incidence of malunion. Patients treated in tubigrip experienced no more pain in weeks 4 and 6 and apart from one failure, no patient actively disliked this form of treatment and 22 were totally satisfied.

Linkage of cystic fibrosis locus and polymorphic DNA markers in 14 families.

Linkage relationships between the cystic fibrosis (CF) locus and three polymorphic DNA markers were examined in 14 families, five of which were of Hispanic origin. Tight linkage was found between the CF locus and MET (maximum lod score = 7.16 at theta = .001), and between CF and pJ3.11 (maximum lod score = 3.87 at theta = .001). We observed two recombinations between CF and collagen, yielding a maximum lod score of 0.359 at theta = .125, and one recombination in the cluster CF-MET-pJ3.11. Analysis by the seriation method indicates the order COL-pJ3.11-CF-MET.

Phosphorylation of tyrosine in the carboxyl-terminal tryptic peptide of pp60c-src.

The major site of tyrosine phosphorylation of the transforming protein of Rous sarcoma virus, pp60v-src (tyrosine-416), is different from the major site of tyrosine phosphorylation of its nontransforming normal cellular counterpart, pp60c-src. We have shown that antibodies against a synthetic peptide modeled on the carboxyl-terminal 13 residues of pp60c-src specifically immunoprecipitate the major phosphotyrosine tryptic peptide of pp60c-src from both chicken and rat fibroblasts. These experiments localize the major site of tyrosine phosphorylation to one or more of the three tyrosine residues in the carboxyl-terminal tryptic peptide at positions 511, 519, and 527 of the amino acid sequence of chicken pp60c-src. Tyrosines-519 and -527 are in the carboxyl-terminal 19-amino acid segment of pp60c-src that is deleted and replaced by an unrelated sequence in pp60v-src. It is possible that phosphorylation of tyrosine in the carboxyl-terminal tryptic peptide may be involved in the normal regulation of pp60c-src. The absence of this phosphorylation site in pp60v-src may, in part, contribute to its oncogenic properties.