The relative involvement of Th1 and Th2 associated immune responses in the expulsion of a primary infection of Heligmosomoides polygyrus in mice of differing response phenotype.

T helper cell (Th1 and Th2) associated responses were examined following a primary infection with the gastrointestinal nematode Heligmosomoides polygyrus in five inbred strains of mice with different resistance phenotypes. Levels of (i) mast cell protease, (ii) specific IgE, (iii) nitric oxide and (iv) specific IgG2a, as markers of Th2 and Th1 associated responses, respectively, were determined in sera and intestinal fluids and correlated with worm burdens. The "fast" responder (resistant) strains SWR and SJL produced strong Th2 and Th1 associated responses respectively in a mutually exclusive fashion. The F1 hybrid (SWRxSJL) F1, showed rapid expulsion of the parasite and expressed both intense Th1 and Th2 responses, suggesting synergism between Th1 and Th2 activity in these mice. The results indicate that both Th2 and Th1 responses operate in mice following a primary infection with H. polygyrus and that each Th response may be involved to a greater or lesser degree within certain strains. Resistance to H. polygyrus was found to correlate only to the intensity of either the gut-associated mastocytosis or nitric oxide production in these strains but not to either specific IgE or IgG2a titres. Chronic infections in the "slow" response phenotype mouse strains CBA and C57BL/10, were associated with both poor Th2 and poor Th1-associated responses attributed to a general parasite-mediated immunosuppression of the host immune response to infection.

Response heterogeneity of human macrophages to ATP is associated with P2X7 receptor expression but not to polymorphisms in the P2RX7 promoter.

A region 2 kb upstream of exon 1 of the P2X7 gene was sequenced using DNA from nine healthy individuals who exhibited three different ATP response phenotypes (i.e. high, low and interferon gamma-inducible). Five single nucleotide polymorphisms were identified within the nine donor promoter sequences but none were associated with a specific ATP response phenotype. A P2X7 loss of function polymorphism (1513 in exon 13) was also screened for within donor DNA but no response associations were identified. ATP response phenotype was positively associated with P2X(7) receptor expression, as assessed by flow cytometry, but not with any identified receptor or promoter gene polymorphisms.

Effect of oxygen radicals and differential expression of catalase and superoxide dismutase in adult Heligmosomoides polygyrus during primary infections in mice with differing response phenotypes.

The ability of oxygen radicals to kill Heligmosomoides polygyrus adult worms was examined by assessing parasite survival following incubation with hydrogen peroxide and acetaldehyde/xanthine oxidase, generators of H2O2 and H2O2/O2(-), respectively. H. polygyrus worms could tolerate levels of < 0.25 mM hydrogen peroxide and < 0.5 mM/20 mU acetaldehyde/xanthine oxidase for 20 h, but, at higher concentrations, marked sex-dependent susceptibility was observed, with males being more sensitive to H2O2 and O2(-) than female worms. The ability to evade free radical-mediated damage was also evaluated by measuring superoxide dismutase (SOD) and catalase levels in worms isolated at different time points from four strains of mice with differing resistance phenotypes. Levels of both catalase and SOD in female worms isolated from 'rapid'[(SWRxSJL)F1], 'fast' (SWR) or 'intermediate' (BALB/c), but not 'slow' (C57BL/10), responder mice showed a strain-dependent increase with time. Moreover, male worms were rejected faster than female worms in the 'rapid', 'fast' and 'intermediate' responder strains of mice. The results suggest that host-derived free radicals can damage adult worms and that female worms can increase production of their scavenging enzymes in response to the immune onslaught that eventually leads to worm expulsion in mice with 'fast', 'rapid' or 'intermediate' response phenotypes.

Interferon-gamma receptor deficiency mimicking Langerhans' cell histiocytosis.

Two patients who were initially given a diagnosis of Langerhans' cell histiocytosis on the basis of the clinical, radiologic, and biopsy findings had mycobacterial infection subsequently identified. The correct diagnosis of dominant partial interferon-gamma receptor deficiency was established.

ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X(7)-dependent process inducing bacterial death by phagosome-lysosome fusion.

Mycobacterium tuberculosis survives within host macrophages by actively inhibiting phagosome fusion with lysosomes. Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular mycobacteria. The following studies were undertaken to characterize the effector pathway(s) involved. Macrophages were obtained from p47(phox) and inducible NO synthase gene-disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals, respectively) and P2X(7) gene-disrupted mice. RAW murine macrophages transfected with either the natural resistance-associated macrophage protein gene 1 (Nramp1)-resistant or Nramp1-susceptible gene were also used. The cells were infected with bacille Calmette-Guérin (BCG), and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy. P2X(7) receptor activation was essential for effective ATP-induced mycobacterial killing, as its bactericidal activity was radically diminished in P2X(7)(-/-) macrophages. ATP-mediated killing of BCG within p47(phox-/-), inducible NO synthase(-/-), and Nramp(s) cells was unaffected, demonstrating that none of these mechanisms have a role in the ATP/P2X(7) effector pathway. Following ATP stimulation, BCG-containing phagosomes rapidly coalesce and fuse with lysosomes. Blocking of macrophage phospholipase D activity with butan-1-ol blocked BCG killing, but not macrophage death. ATP stimulates phagosome-lysosome fusion with concomitant mycobacterial death via P2X(7) receptor activation. Macrophage death and mycobacterial killing induced by the ATP/P2X(7) signaling pathway can be uncoupled, and diverge proximal to phospholipase D activation.

Impaired interferon gamma-mediated immunity and susceptibility to mycobacterial infection in childhood.

Mendelian susceptibility to poorly virulent mycobacteria such as bacillus Calmette-Guerin (BCG) and environmental nontuberculous mycobacteria is a clinically heterogeneous syndrome. The clinical features of affected children cover a continuous spectrum from disseminated lethal bacillus Calmette-Guerin infection to local recurrent nontuberculous mycobacterial infection. Different types of mutations in four genes (IFNGR1, IFNGR2, IL12B, IL12RB1) have revealed both allelic and nonallelic heterogeneity and result in eight different disorders whose common pathogenic pathway is impaired interferon gamma (IFNgamma) mediated immunity. The severity of the clinical phenotype depends on the genotype. Complete IL-12 p40 and IL-12 receptor beta1 deficiencies and partial IFNgamma receptor 1 (IFNgammaR1) and IFNgammaR2 deficiencies generally lead to curable infections at various ages, and antibiotics supplemented with IFNgamma if required are likely to be effective. Complete IFNgammaR1 and IFNgammaR2 deficiencies predispose to overwhelming infection in early childhood, which may respond to antibiotics but relapse when antibiotics are discontinued. Rapid discrimination between complete IFNgammaR1 and IFNgammaR2 deficiency and other defects, therefore, is an important diagnostic step for planning clinical management.

ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guérin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes.

We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected human macrophages. ATP increases the cytosolic Ca(2+) concentration in macrophages by mobilizing intracellular Ca(2+) via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca(2+) via P2X purinoceptors. The relative contribution of these receptors and Ca(2+) sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca(2+) in UTP-desensitized macrophages (in Ca(2+)-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca(2+) store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca(2+)-mobilizing P2Y purinoceptor in addition to the P2Y(2) subtype is expressed on human macrophages. ATP and the Ca(2+) ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca(2+)-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid acetoxymethyl ester (an intracellular Ca(2+) chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca(2+) mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.

Diagnosis of defects in the type 1 cytokine pathway.

Patients with inherited defects in the interleukin-12 (IL-12)-dependent, 'high-output' interferon-gamma (IFN-gamma) pathway exhibit selective susceptibility to poorly pathogenic mycobacterial and salmonella infections. This review summarises the extended clinical spectrum seen in this group of patients and indicates a strategy for the identification of putative defects in the type 1 cytokine pathway.

Residual type 1 immunity in patients genetically deficient for interleukin 12 receptor beta1 (IL-12Rbeta1): evidence for an IL-12Rbeta1-independent pathway of IL-12 responsiveness in human T cells.

Genetic lack of interleukin 12 receptor beta1 (IL-12Rbeta1) surface expression predisposes to severe infections by poorly pathogenic mycobacteria or Salmonella and causes strongly decreased, but not completely abrogated, interferon (IFN)-gamma production. To study IL-12Rbeta1-independent residual IFN-gamma production, we have generated mycobacterium-specific T cell clones (TCCs) from IL-12Rbeta1-deficient individuals. All TCCs displayed a T helper type 1 phenotype and the majority responded to IL-12 by increased IFN-gamma production and proliferative responses upon activation. This response to IL-12 could be further augmented by exogenous IL-18. IL-12Rbeta2 was found to be normally expressed in the absence of IL-12Rbeta1, and could be upregulated by IFN-alpha. Expression of IL-12Rbeta2 alone, however, was insufficient to induce signal transducer and activator of transcription (Stat)4 activation in response to IL-12, whereas IFN-alpha/IFN-alphaR ligation resulted in Stat4 activation in both control and IL-12Rbeta1-deficient cells. IL-12 failed to upregulate cell surface expression of IL-18R, integrin alpha6, and IL-12Rbeta2 on IL-12Rbeta1-deficient cells, whereas this was normal on control cells. IL-12-induced IFN-gamma production in IL-12Rbeta1-deficient T cells could be inhibited by the p38 mitogen-activated protein kinase (MAP) kinase inhibitor SB203580 and the MAP kinase kinase (MEK) 1/2 inhibitor U0126, suggesting involvement of MAP kinases in this alternative, Stat4-independent, IL-12 signaling pathway.Collectively, these results indicate that IL-12 acts as a partial agonist in the absence of IL-12Rbeta1. Moreover, the results reveal the presence of a novel IL-12Rbeta1/Stat4-independent pathway of IL-12 responsiveness in activated human T cells involving MAP kinases. This pathway is likely to play a role in the residual type 1 immunity in IL-12Rbeta1 deficiency.

The relationship between circulating and intestinal Heligmosomoides polygyrus-specific IgG1 and IgA and resistance to primary infection.

Specific serum and intestinal immunoglobulin (Ig)G1 and IgA responses to Heligmosomoides polygyrus were measured in a panel of seven inbred mouse strains which exhibit 'rapid' (<6 weeks (SWRxSJL)F1), 'fast' (<8 weeks, SJL and SWR), 'intermediate' (10-20 weeks, NIH and BALB/c) or 'slow' (>25 weeks, C57BL/10 and CBA) resolution of primary infections. Mice with 'rapid', 'fast' or 'intermediate' response phenotypes produced greater serum and intestinal antibody responses than those with 'slow' phenotypes. The F1 hybrids ((SWRxSJL)F1) of two 'fast' responder strains showed the earliest antibody response with maximum titres evident within 6 weeks of infection. There was a negative correlation between the serum IgG1 responses and worm burdens in individual mice within a number of mouse strains, and also between serum IgG1 and IgA responses and worm burdens in the 'rapid' ((SWRxSJL)F1) responder strain. The presence of IgG1 in the gut was found to be due to local secretion rather than plasma leakage. Using Western immunoblotting, serum IgG1 from 'rapid' and 'fast' responder but not 'slow' responder mice was found to react with low molecular weight antigens (16-18 kDa) in adult worm excretory/secretory products.

A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection.

The immunogenetic basis of severe infections caused by bacille Calmette-Guérin vaccine and environmental mycobacteria in humans remains largely unknown. We describe 18 patients from several generations of 12 unrelated families who were heterozygous for 1 to 5 overlapping IFNGR1 frameshift small deletions and a wild-type IFNGR1 allele. There were 12 independent mutation events at a single mutation site, defining a small deletion hotspot. Neighbouring sequence analysis favours a small deletion model of slipped mispairing events during replication. The mutant alleles encode cell-surface IFNgamma receptors that lack the intra-cytoplasmic domain, which, through a combination of impaired recycling, abrogated signalling and normal binding to IFNgamma exert a dominant-negative effect. We thus report a hotspot for human IFNGR1 small deletions that confer dominant susceptibility to infections caused by poorly virulent mycobacteria.

Inherited interleukin 12 deficiency in a child with bacille Calmette-Guérin and Salmonella enteritidis disseminated infection.

Interferon-gamma receptor ligand-binding chain (IFN-gammaR1) or signaling chain (IFN-gammaR2) deficiency, like interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency, predispose to severe infections due to poorly virulent mycobacteria and salmonella. A child with bacille Calmette-Guérin and Salmonella enteritidis infection was investigated. Mutations in the genes for IFN-gammaR1, IFN-gammaR2, IL-12Rbeta1, and other molecules implicated in IL-12- or IFN-gamma-mediated immunity were sought. A large homozygous deletion within the IL-12 p40 subunit gene was found, precluding expression of functional IL-12 p70 cytokine by activated dendritic cells and phagocytes. As a result, IFN-gamma production by lymphocytes was markedly impaired. This is the first discovered human disease resulting from a cytokine gene defect. It suggests that IL-12 is essential to and appears specific for protective immunity to intracellular bacteria such as mycobacteria and salmonella.

Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency.

In humans, interferon gamma (IFN-gamma) receptor deficiency leads to a predisposition to mycobacterial infections and impairs the formation of mature granulomas. Interleukin-12 (IL-12) receptor deficiency was found in otherwise healthy individuals with mycobacterial infections. Mature granulomas were seen, surrounded by T cells and centered with epithelioid and multinucleated giant cells, yet reduced IFN-gamma concentrations were found to be secreted by activated natural killer and T cells. Thus, IL-12-dependent IFN-gamma secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12-independent IFN-gamma secretion.

ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z(P2X7) receptors.

The death of BCG-infected human macrophages induced in vitro by ligation of surface CD95 (Fas), CD69, or complement-mediated lysis was shown not to result in the death of intracellular mycobacteria, whereas exposure to extracellular ATP initiated both macrophage death and killed the intracellular bacteria. ATP acted via P2Z receptors because these effects were mimicked by benzoylbenzoic ATP (a known agonist of P2Z receptors) and blocked by oxidized ATP, DIDS, suramin, amiloride, and KN62 (known inhibitors of P2Z-mediated responses). ATP-mediated bacterial killing was independent of reactive nitrogen and oxygen intermediates and of actinomycin D or cycloheximide inhibition. ATP-induced macrophage cell death, BCG killing, and lucifer yellow dye incorporation were minimal in 2 out of 19 healthy donors. The results suggest possible genetic heterogeneity of this mechanism of mycobacterial killing associated with P2Z-mediated pore formation.

Simple method for pretreatment of tissue sections for the detection of apoptosis by in situ end-labelling and in situ nick translation.

Aims-To overcome the problems associated with proteolytic pretreatment of tissue sections for the detection of apoptosis.Methods-Formalin fixed, paraffin wax embedded tissue sections of reactive lymph nodes and biopsy specimens of Burkitt lymphoma were pretreated by pressure cooking for the detection of apoptosis using the in situ end-labelling and in situ nick translation methods.Results-The results achieved with the in situ end-labelling and nick translations methods were compared with those obtained using a novel anti-apoptosis specific protein (ASP) antibody. The staining patterns generated using the three methods were similar and consistent, although the ASP antibody seemed to be more sensitive and detected higher numbers of apoptotic cells within sections.Conclusions-Pressure cooking is advocated as an alternative method to proteolytic enzyme digestion for pretreating paraffin wax sections. It is reliable, inexpensive, reduces the need to optimise pretreatment variables for different tissues, and permits double immunostaining of sections.

CD4+ cytolytic T cells can destroy autologous and MHC-matched macrophages but fail to kill intracellular Mycobacterium bovis-BCG.

Mycobacterium bovis-BCG infected macrophages were exposed in vitro to PPD-stimulated T lymphocytes from tuberculin responsive donors or to a panel of mycobacterial-antigen specific CD4+ T cell clones. Both polyclonal and clonal T cells caused considerable antigen-specific lysis of autologous or MHC class II matched macrophages. However, lysis of infected macrophages did not significantly affect the number of viable mycobacteria which were released into the culture media from lysed macrophages. In tuberculosis, CD4+ cytolytic T cells may be primarily involved in tissue destruction and lack a significant role in acquired cellular immunity.

Lysis of human macrophages by cytolytic CD4+ T cells fails to affect survival of intracellular Mycobacterium bovis-bacille Calmette-Guérin (BCG).

Human CD4+, mycobacteria-specific, cytolytic T cell clones were used to lyse BCG-infected macrophages, and the effect on the subsequent growth and viability of the organisms was examined. The survival of released bacteria following cell lysis was assessed by both 3H-uridine labelling and colony-forming unit (CFU) estimation. The results indicate that even when effective antigen-specific or lectin-mediated cytolysis of the infected macrophages was achieved, there was no evidence for a direct mycobactericidal effect on the intracellular bacteria. This remained the case even if the period of co-culture of T cells and macrophages was extended up to 48 h. Pretreatment of the macrophages with interferon-gamma (IFN-gamma) was not able to act together with T cell-mediated lysis to produce inhibition of mycobacterial growth.

Effect of blocking TNF-alpha on intracellular BCG (Bacillus Calmette Guerin) growth in human monocyte-derived macrophages.

Four agents, thalidomide, oxpentifylline, dexamethasone and a polyclonal anti-TNF-alpha antibody, were all shown by specific Elisa to block endogenous TNF-alpha production by Bacillus Calmette Guerin (BCG)-infected human monocyte-derived macrophages in in vitro culture. There was however no significant enhancement of intracellular BCG growth, over a 7-day incubation, in human monocyte-derived macrophages in the presence of any of the TNF-alpha-blocking agents, as determined by both radiometric and CFU counting methods of assessing bacterial viability and growth. The result suggests that the action of TNF-alpha alone is unlikely to be an important effector mechanism in antimycobacterial immunity within human cells.