The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene.

In mice, natural resistance or susceptibility to infection with intracellular parasites is determined by a locus or group of loci on chromosome 1, designated Bcg, Lsh, and Ity, which controls early microbial replication in reticuloendothelial organs. We have identified by positional cloning a candidate gene for Bcg, Nramp1, which codes for a novel macrophage-specific membrane transport protein. We have created a mouse mutant bearing a null allele at Nramp1, and we have analyzed the effect of such a mutation on natural resistance to infection. Targeted disruption of Nramp1 has pleiotropic effects on natural resistance to infection with intracellular parasites, as it eliminated resistance to Mycobacterium bovis, Leishmania donovani, and lethal Salmonella typhimurium infection, establishing that Nramp1, Bcg, Lsh, and Ity are the same locus. Comparing the profiles of parasite replication in control and Nramp1-/- mice indicated that the Nramp1Asp169 allele of BcgS inbred strains is a null allele, pointing to a critical role of this residue in the mechanism of action of the protein. Despite their inability to control parasite growth in the early nonimmune phase of the infection, Nramp1-/- mutants can overcome the infection in the late immune phase, suggesting that Nramp1 plays a key role only in the early part of the macrophage-parasite interaction and may function by a cytocidal or cytostatic mechanism distinct from those expressed by activated macrophages.

Human natural resistance-associated macrophage protein: cDNA cloning, chromosomal mapping, genomic organization, and tissue-specific expression.

Natural resistance to infection with unrelated intracellular parasites such as Mycobacteria, Salmonella, and Leishmania is controlled in the mouse by a single gene on chromosome 1, designated Bcg, Ity, or Lsh. A candidate gene for Bcg, designated natural resistance-associated macrophage protein (Nramp), has been isolated and shown to encode a novel macrophage-specific membrane protein, which is altered in susceptible animals. We have cloned and characterized cDNA clones corresponding to the human NRAMP gene. Nucleotide and predicted amino acid sequence analyses indicate that the human NRAMP polypeptide encodes a 550-amino acid residue membrane protein with 10-12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionary conserved consensus transport motif. Identification of genomic clones corresponding to human NRAMP indicates that the gene maps to chromosome 2q35 within a group of syntenic loci conserved with proximal mouse 1. The gene is composed of at least 15 exons, with several exons encoding discrete predicted structural domains of the protein. These studies have also identified an alternatively spliced exon encoded by an Alu element present within intron 4. Although this novel exon was found expressed in vivo, it would introduce a termination codon in the downstream exon V, resulting in a severely truncated protein. Northern blot analyses indicate that NRAMP mRNA expression is tightly controlled in a tissue-specific fashion, with the highest sites of expression being peripheral blood leukocytes, lungs, and spleen. Additional RNA expression studies in cultured cells identified the macrophage as a site of expression of human NRAMP and indicated that increased expression was correlated with an advanced state of differentiation of this lineage.

Genetic control of host resistance to infection.

Human resistance to infectious diseases is often regulated by multiple genes that control different aspects of host-parasite interaction. Genetically distinct inbred strains of mice that differ in their susceptibility to specific pathogens are invaluable for dissecting such complex patterns and have allowed the identification of several host-resistance loci that regulate natural and acquired immunity in response to infection. Cloning these genes is the first step in elucidating their roles in host defense.

Genetics of the immune response to infectious pathogens.

In this selective review of the recent literature in the field of genetically determined host resistance to infection, we highlight five areas in which research is directed towards the search for proteins encoded by genes that function to maintain a 'resistant' phenotype in the face of challenge by a variety of pathogenic organisms. In particular, we discuss newly described genes that may regulate host resistance, newly described functions of genes previously identified, the reverse genetics approach to cloning an elusive gene, a direct genetics approach to a similar problem, and the role of the major histocompatibility complex in regulating our ability to resist challenge by infectious organisms.

Immunogenetics in the analysis of resistance to intracellular pathogens.

Recent studies have identified genes involved in resistance to intracellular pathogens. Such genes include the murine MHC class I gene, Ld (toxoplasmosis), HLA-BW53, HLA DRB1* 1302-DQ B10s01 and TNF2 (malaria), murine Nramp (toxoplasmosis, leishmaniasis and tuberculosis), gene(s) modulating the T-helper type 1 and type 2 dichotomy (leishmaniasis, leprosy and HIV infection) and the natural killer cell complex (cytomegalovirus infection). There also have been other advances in immunogenetics that have led to a better understanding of resistance to intracellular pathogens. These include effector mechanisms of immune response genes and factors modulating genetic susceptibility. Identification of genes that determine resistance/susceptibility (and their effector mechanisms) has impacted on vaccine development. Immunogenetics has been important in characterizing roles of TCR genes, superantigens, and host genes that play a role in molecular mimicry in disease pathogenesis. In addition, recent work with gene knockout, recombinant inbred or congenic, mutant, consomic, and transgenic mice, positional cloning, mouse/human gene homologies to identify candidate human resistance genes, and the rapid expansion of the gene transcription maps of the human genome, have been important in analysis of resistance to intracellular pathogens.

Two distinct regions in the 3' untranslated region of tumor necrosis factor alpha mRNA form complexes with macrophage proteins.

The production of tumor necrosis factor alpha (TNF-alpha), a key proinflammatory cytokine essential for the function of the immune system, is regulated at both the transcriptional and posttranscriptional levels. In this report, we focus on the interaction of TNF-alpha mRNA with macrophage proteins, likely mediators of its post-transcriptional control. Mapping of murine TNF-alpha mRNA by using a combination of RNase protection and RNA gel shift assays revealed that two distinct sites within the 3' untranslated region (3'-UTR) engage in the formation of four major RNA-protein complexes, while no protein binding to the 5'-UTR or coding sequences was detected. The protein-binding site of three RNA-protein complexes, A, B, and C, is positioned between bases 1291 and 1320 inside the AU-rich sequence, a region previously shown to be crucial for both translational repression and lipopolysaccharide inducibility of TNF-alpha. An additional protein complex (complex D) whose binding to the TNF-alpha 3'-UTR was independent of the presence of AU-rich sequences was identified. At least six protein species with apparent molecular masses of 48, 52, 54, 81, 101, and 150 kDa are in direct contact with TNF-alpha mRNA. The RNA-binding proteins are differentially distributed in the cell: complexes A and D are present predominantly in the cytosol, while complexes B and C are found in the nucleus and associated with particulate cytoplasmic fractions. Cytosolic complex A displays comparatively high specificity for TNF-alpha mRNA, while the binding of complexes B and C to TNF-alpha mRNA is readily competed for by other AU-rich sequence-containing RNAs. In summary, these findings demonstrate that two regions of the TNF-alpha mRNA molecule interact with macrophage RNA-binding protein complexes that differ in their core protein composition, cellular distribution, and affinity to TNF-alpha mRNA.

Toll-like receptors, cytokines and the immunotherapeutics of asthma.

Asthma is a complex disease caused by a poorly characterized set of genetic and environmental factors whose pathology is a result of immune dysregulation. Toll-like receptors are pathogen associated molecular pattern receptors expressed by many airway and pulmonary tissues as well as cells of the innate and adaptive immune system. Ligation of toll-like receptors can lead to a change in the expression levels of multiple inflammatory and anti-inflammatory mediators which are involved in the pathogenesis of asthma. These ligands and their receptors are therefore prime candidates in the search for immunotherapeutic treatments of asthma. The use of murine models of allergic asthma as tools for the genetic dissection of this disease should allow the molecular mechanisms underlying asthma to be identified and possibly used as further immunotherapeutic targets.

Susceptibility to urethan-induced pulmonary adenomas between A/J and C57BL/6J mice: use of AXB and BXA recombinant inbred lines indicating a three-locus genetic model.

For insight into the number of genes governing differential susceptibility such as in mice of the inbred strain A/J (A) highly susceptible to the induction of pulmonary adenomas by urethan in comparison to resistant strain C57BL/6J (B6) mice, tumor induction was studied in the AXB and BXA series of recombinant inbred (RI) strains derived respectively from A female X B6 male and B6 female X A male ancestral lines. Mice from 46 of these lines were given injections with 1 mg urethan/g (body wt), and the tumor number was assessed 4 months later. Lung tumor multiplicity for several RI lines was independently characterized in the Boulder, CO, and Montreal laboratories, and very similar numbers were obtained. Most lines had multiplicities intermediate to those of the progenitor strains, clearly indicating that more than a single gene accounted for the differences in lung tumor susceptibility between A and B6. The tumor incidence and multiplicity data fit very closely to what would be expected under a three-locus model, and we designated these genes "Pas" for pulmonary adenoma susceptibility. One locus called "Pas-1" had an effect on tumor multiplicity greater than that of the other loci.

Haplotype-specific interactions of non-H-2-linked genetic factors controlling the mouse C4 and Slp protein levels.

The influence of non-H-2 linked genes on the plasma levels of the H-2 S-region encoded proteins C4, Slp, and factor B was tested in Recombinant Inbred (RI) strains. The A X B and B X A RI strains exhibit a continuous range of C4 and Slp levels from very high to very low which reach beyond the levels of their parental strains, C57BL/6J and A/J, indicating involvement of several trans-regulatory (non-H-2-linked) genes. Only limited variation in levels of factor B has been found. No linkage relationship could be established for the trans-regulatory genes, because more than one gene is involved. A complex interaction of H-2 haplotype, genetic background, sex, and possibly maternal effect in determining the C4 and Slp protein plasma levels has been observed. The H-2-dependent sex effect is evident, because males have higher C4 levels than females in RI strains with H-2b but not with H-2a haplotype. This sex effect is also background dependent, because it is present in the H-2b congenic strain on A background (A.BY) but not in C57BL/10 and C57BL/6 (both H-2b). Mice from RI strains with H-2b haplotype have in general higher C4 levels than mice with H-2a haplotype.

Recombinant inbred mouse strains derived from A/J and C57BL/6J: a tool for the study of genetic mechanisms in host resistance to infection and malignancy.

A number of recombinant inbred mouse strains (currently 48) have been developed from the strains A/J and C57BL/6J. These strains are likely to become an excellent probe in the study of genetic mechanisms underlying host resistance to infections and tumors.

Amyloid-enhancing factor: production and response in amyloidosis-susceptible and -resistant mouse strains.

Genetic variations in the development of casein-induced amyloidosis exist among inbred strains of mice: CBA/J and C57BL/6J mice are susceptible, while A/J strain mice are resistant to this disease. Amyloidosis is usually induced by daily injections of an inflammatory stimulus for 2-3 wk. The deposition of amyloid in experimental animals can be accelerated by injection of a material called amyloid-enhancing factor (AEF); when injected concomitantly with an inflammatory stimulus, AEF provokes appearance of amyloidosis as early as 2 days after injection. AEF is extracted from amyloid laden or from normal organs (although in small amount). Our studies were designed to determine if the resistance to amyloidosis seen in A/J mice was either due to a lack of AEF production or to an inability of these mice to respond to AEF. A standard source of CBA/J-derived AEF facilitated the development of amyloidosis in the organs of both the susceptible (CBA/J, C57BL/6J) and the resistant A/J mice. On the contrary, amyloidosis was only induced in susceptible CBA/J hosts when material derived from susceptible (CBA/J, C57BL/6J) animals was injected. CBA/J mice injected with A/J-derived AEF preparation did not develop amyloidosis. These results thus suggest that the determination of resistance or susceptibility to secondary amyloidosis could operate at the level of AEF production.

Natural resistance to infection with intracellular parasites: molecular genetics identifies Nramp1 as the Bcg/Ity/Lsh locus.

Natural resistance to infection with intracellular parasites is controlled in the mouse by the expression of a locus or group of loci on chromosome 1 alternatively named Bcg, Lsh, and Ity. Bcg affects the capacity of mature tissue macrophages to restrict the intracellular proliferation of ingested parasites in the reticuloendothelial organs of the host during the early phase of infection. This review summarizes our molecular genetic approach to the isolation and characterization of the Bcg locus. We have used a positional cloning strategy based on genetic and physical mapping, YAC cloning, and exon trapping to isolate a candidate gene for Bcg, named Nramp1, which codes for a macrophage-specific polytopic protein with 12 predicted transmembrane domains and a consensus transport motif. Sequence analysis of Nramp1 cDNA clones from 27 Bcgs and Bcgr mouse strains reveals that susceptibility to infection (Bcgs) is associated with a single nonconservative Gly to Asp substitution at position 169 within predicted transmembrane domain 4 of the Nramp protein. Cloning experiments and homology search in available databases demonstrated that the Nramp1 gene belongs to a small gene family with several members in vertebrates and in such distantly related species as yeast and plants. Nramp proteins share a remarkable degree of similarity, with strong amino acid sequence conservation in the transmembrane domains, suggesting a common transport function for the Nramp family. Finally, we generated Nramp1-/- gene knockout mice, and analysis of their phenotypic characteristics established that (1) Nramp1 plays a key role in natural defense against infection with intracellular parasites and therefore demonstrated allelism between Nramp1 and Bcg/Ity/Lsh, (2) Nramp1 functions by a novel cytocidal/cytostatic mechanism distinct from those expressed by the activated macrophage, and (3) the Nramp1Asp169 allele of Bcgs inbred strains is a null allele, pointing to a critical role of this residue in Nramp1 function.

Quantitative model of mononuclear phagocyte lineage proliferation in murine bone marrow.

An analysis and a synthesis of published and original experimental evidence have been performed with a view to providing a global qualitative and quantitative description of the proliferation of monocyte precursors in murine bone marrow. Two versions of a mathematical model are proposed. The first consists of two dividing generations of granulocyte-macrophage colony-forming cells (GM-CFC) and two dividing generations of macrophage colony-forming cells (M-CFC), differentiating into promonocytes (PM) and then into monocytes (MC). The second version consists of two generations of GM-CFC and three generations of M-CFC, followed by PM and MC. Both are compatible with experimental data, and further evidence is needed to elaborate a unique representation. The kinetic constants have been taken from published material whenever possible and estimated from available data and model kinetics in the case of unknown quantities. The models are applied to a pulse labeling assay, and calculated labeled subpopulations are compared with experimental values. Agreement is satisfactory for both versions in the case of MC, while a calculated PM labeling index falls more rapidly than the experimental values. However, inconsistency also exists between PM and MC experimental data.

Genetic control of macrophage activation for killing of extracellular targets.

Activated peritoneal macrophages from Corynebacterium parvum-treated mice of most inbred strains, including C57BL/6J (B), are cytotoxic to adherent 1023 sarcoma target cells as well as to larvae of Schistosoma mansoni. Macrophages from A/J (A) strain mice, on the other hand, are defective in this function. Segregation analysis of these two traits was compatible with the hypothesis that effective activation is, in each case, controlled by a single, dominant, autosomal gene. Typing of individual animals of the segregating backcross progeny and of AXB/BXA recombinant inbred strains for the expression of macrophage activation for tumoricidal and schistosomulicidal activity indicates that the genetic control of these two traits is closely linked or identical.

Killing of Mycobacterium smegmatis by macrophages from genetically susceptible and resistant mice.

The bactericidal function of macrophages was investigated in congenic mice expressing the phenotype of susceptibility (B10.A, Bcgs) or resistance (B10.ABcgr) to mycobacterial infection. When splenic and peritoneal macrophages from these two mouse strains were infected in vitro with Mycobacterium smegmatis, the Bcgr macrophages were shown to inactivate M. smegmatis more efficiently than their Bcgs congenic counterparts. The mechanisms of this superior antimycobacterial activity was studied further. Addition of catalase did not abolish killing to a significant degree in either allelic type of macrophage, suggesting that hydrogen peroxide production was not involved in the killing activity controlled by the Bcg gene. Activation of Bcgs macrophages by exposure to crude lymphokines rendered them equally as efficient as their Bcgr counterparts in their capacity to destroy M. smegmatis. This finding suggests that both the genetically resistant and susceptible macrophages have the potential to kill M. smegmatis in vitro. This potential is expressed constitutively by the Bcgr but not Bcgs macrophages and can be induced, by lymphokine treatment, in the Bcgs macrophages. In a final set of experiments, the macrophage killing of M. smegmatis was evaluated as a test system to type for the Bcg gene allelic type in vitro, using a set of AXB and BXA recombinant inbred strains of mice. Results obtained show that typing of AXB/BXA recombinant inbred strains for the trait of bactericidal activity vs. M. smegmatis in vitro revealed a perfect match with the strain distribution pattern of resistance/susceptibility to Mycobacterium bovis BCG in vivo.

Genetic resistance/susceptibility to mycobacteria: phenotypic expression in bone marrow derived macrophage lines.

Congenic strains of mice susceptible (B10A.Bcgs) or resistant (B10A.Bcgr) to BCG were established. Here we describe the model system which has been established to analyze the functional activities of macrophages in the two strains. We have immortalized bone marrow macrophages from B10A.Bcgs and B10A.Bcgr congenic strains of mice and derived cloned macrophage lines designated B10S and B10R, respectively. B10R and B10S cell lines exhibited surface markers and morphology typical of macrophages. B10S and B10R were similar in their phagocytic activity, in their level of c-fms, in their transforming growth factor beta (TGF beta) mRNAs expression, and in their expression of tumoricidal activity in response to interferon-gamma (IFN gamma) plus lipopolysaccharides (LPS). However, B10R macrophages expressed a higher level of la mRNA when activated with IFN gamma compared with B10S macrophages. Analysis of the bacteriostatic activity of the two cell lines revealed that B10R macrophages were much more active in inhibiting Mycobacterium smegmatis replication than B10S. To measure the intracellular destruction of bacilli, a bactericidal assay based on hybridization with an oligonucleotide probe specific for mycobacterial ribosomal RNA was designed. The results demonstrated that B10R macrophages were endowed with enhanced constitutive bactericidal activity as compared with B10S. In conclusion we have obtained macrophage lines from bone marrow of B10A.Bcgs and B10A.Bcgr mice that express to a similar extent functional and phenotypic characteristics of macrophages. However, we demonstrate that relative to B10S macrophages, the B10R macrophages have higher expression of la mRNA and that they are constitutively more active in expressing mycobactericidal activity.

I-A beta gene expression regulation in macrophages derived from mice susceptible or resistant to infection with M. bovis BCG.

The innate capacity of mice to control mycobacterial multiplication early after infection is controlled by the resistant allele of the Nramp-1/Bcg gene. The Bcg gene seems to be involved in a pathway leading to macrophage activation. It differentially affects the ability of BCG-resistant and -susceptible strains of mice to express important macrophage genes including Major Histocompatibility Complex (MHC) class II genes. An inhibition of Nramp1 gene by Nramp1-ribozyme transfection in macrophages resulted in the impairment of MHC class II gene induction by IFN gamma. In this study, we have investigated the molecular mechanisms involved in IFN-gamma-induced MHC class II expression using macrophages derived from mice resistant or susceptible to mycobacterial infections (B10R and B10S, respectively). We have found that the difference in the IFN gamma-induced Ia surface protein expression between B10R and B10S macrophages correlate with a higher rate of I-A beta gene transcription. We have also studied the binding of proteins prepared from nuclear extracts of non-stimulated and IFN-gamma-stimulated B10R and B10S macrophages to the S, X and Y cis-acting elements of the I-A beta promoter. Differences observed in protein binding to the X box may explain the difference in transcription activation of the I-A beta gene. We have also found that I-A alpha and I-A beta mRNA half-lives measured in IFN gamma-stimulated cells are significantly longer in B10R, compared to B10S macrophages. Overall, our data suggest that both transcriptional and posttranscriptional regulatory mechanisms are responsible for the more efficient expression of I-A beta gene in macrophages carrying a resistant allele of Nramp1 gene.

Liposomal encapsulation of 3'-azido-3'-deoxythymidine (AZT) results in decreased bone marrow toxicity and enhanced activity against murine AIDS-induced immunosuppression.

The effect of liposome encapsulation on the bone marrow toxicity and antiviral activity of AZT in C57BL/6 mice was determined. Liposomal encapsulation of AZT enhanced localization in the liver, spleen, and lung, and reduced localization in bone marrow. AZT administered i.v. (0.08-50 mg/kg/day) had significant bone marrow toxicity (30-50% reduction in cellularity) after five injections, maximum toxicity occurring at greater than or equal to 2 mg/kg/day. Parallel reductions in the number of erythrocytes and leukocytes were observed. AZT encapsulated in liposomes had no bone marrow toxicity at doses of 0.08-10 mg/kg/day, and erythrocyte and leukocyte numbers remained normal. Infection of C57BL/6 mice with LP-BM5 murine leukemia virus suppressed T- and B-cell mitogenic responses. Treatment of LP-BM5 retrovirus-infected mice with 2 mg/kg AZT three times weekly partially protected the mitogenic response at 4 but not at 7 weeks postinfection. Treatment with liposomal AZT resulted in normal T- and B-cell mitogenic responses at both 4 and 7 weeks postinfection.

Assessment of lymphokine profiles in activated lymphocytes by semiquantitative PCR.

Several methods are currently used to detect the expression of specific mRNAs in leukocytes. While Northern blot analysis and RNase protection assays are commonly chosen for quantitative assessment of mRNA levels, these methods require a significant quantity of RNA, making their use unfeasible when limiting numbers of cells are available. Alternatively, use of the reverse transcription-polymerase chain reaction (RT-PCR) technique allows detection of specific mRNAs even at low copy number. It is, however, difficult to establish the conditions which allow consistent semi-quantitative assessment of specific mRNA expression, using the RT-PCR method. We report here a modification of the RT-PCR technique, which has enabled us to compare lymphokine mRNA expression profiles in mixed cell populations activated either in vivo or in vitro. This modification is based on the use of standard RNAs generated by in vitro transcription of size-modified CD3 and IFN-gamma-specific PCR products subcloned into the pGEM3 plasmid. Equal amounts of standard RNAs are introduced into each sample, reverse transcribed and co-amplified with cellular mRNA to control the reproducibility and efficiency of the method. The template therefore follows the cellular RNA through all steps of the analysis, and the corresponding 32P-labelled PCR products are subsequently separated by PAGE procedure. The amount of radioactivity incorporated into lymphokine-specific bands is determined by densitometry and normalized against the density of standard bands. Under optimal PCR conditions this method is linear over a 50-fold range of dilutions. The technique is specific, reproducible and fast, allowing an analysis of lymphokine-specific mRNA profiles in samples containing 10(4)-10(6) cells.