Immune response in mice that lack the interferon-gamma receptor

Interferon-gamma (IFN-gamma) exerts pleiotropic effects, including antiviral activity, stimulation of macrophages and natural killer cells, and increased expression of major histocompatibility complex antigens. Mice without the IFN-gamma receptor had no overt anomalies, and their immune system appeared to develop normally. However, mutant mice had a defective natural resistance, they had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. Immunoglobulin isotype analysis revealed that IFN-gamma is necessary for a normal antigen-specific immunoglobulin G2a response. These mutant mice offer the possibility for the further elucidation of IFN-gamma-mediated functions by transgenic cell- or tissue-specific reconstitution of a functional receptor.     

Inhibitory activity of IL-1 receptor antagonist depends on the balance between binding capacity for IL-1 receptor type 1 and IL-1 receptor type II

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.     

Phenotypic and functional characterization of porcine T-lymphocytes

Monoclonal antibodies (mAb) against leucocyte differentiation antigens have altered the way in which immunologists examine the immune system. These mAb allow to identify distinct surface molecules on leukocyte populations, by which these cells can be classified, isolated and studied for their functional properties. This review summarises the knowledge about differentiation antigens useful in the characterisation of porcine T-lymphocytes. Furthermore it focuses on several properties of porcine T-lymphocytes and T-lymphocyte subpopulations.     

Signal transduction by the IL-1 type I receptor: evidence for the involvement of a receptor-coupled protein kinase

A novel serine/threonine specific protein kinase was found to be associated with the type I IL-1 receptor in the murine T cell lines D10N and EL-4. This kinase was identified in immunoprecipitates from IL-1 stimulated T-cells by its ability to phosphorylate exogenous substrates in the presence of radiolabeled ATP. An endogenous protein, most likely a member of the IL-1 R1 complex, was also phosphorylated. The activation of the kinase is specific for IL-1, neither TNF nor phorbol esters were able to activate the IL-1 RI associated kinase activity. The IL-1 receptor antagonist had no intrinsic activity and inhibited the activation of the kinase. The activation of the kinase was rapid and detectable after 30 seconds of IL-1 stimulation. A minimal model of the IL-RI signal transduction complex is discussed, presenting this novel serine/threonine kinase as a constituent of the complex.     

The type II IL-1 receptor interacts with the IL-1 receptor accessory protein: a novel mechanism of regulation of IL-1 responsiveness

IL-1 binds to two types of receptors on the cell membrane, of which only type I (IL-1RI) transduces signals in concert with the coreceptor IL-1 receptor accessory protein (IL-1RAcP) while type II (IL-1RII) allegedly functions solely as ligand sink and decoy receptor without participating in IL-1 signaling. To investigate the regulatory role of IL-1RII on IL-1 responsiveness, a chimeric receptor encompassing the extracellular and transmembrane portions of IL-1RII and the cytoplasmic signal-transducing domain of IL-1RI was transfected into two murine EL-4-derived sublines that do or do not express IL-1RAcP, respectively. The chimeric receptor was able to transduce the IL-1 signal and induce IL-2 production only in the cell line which expressed IL-1RAcP, suggesting effective interaction between the extracellular domains of IL-1RII and IL-1RAcP in the presence of IL-1. The physical association of ligated IL-1RII with IL-1RAcP was proven by crosslinking experiments with radio-iodinated IL-1 and subsequent immunoprecipitations in normal human B cells and in EL-4 D6/76 cells transiently cotransfected with IL-1RII and IL-1RAcP, respectively. Based on these findings, it is proposed that upon IL-1 binding IL-1RII can recruit IL-1RAcP into a nonfunctional trimeric complex and thus modulate IL-1 signaling by subtracting the coreceptor molecule from the signaling IL-1RI. In this novel mechanism of coreceptor competition, the ratio between IL-1RII and IL-1RI becomes the central factor in determining the IL-1 responsiveness of a cell and the availability of IL-1RAcP becomes limiting for effective IL-1 signaling.     

Expression and functional activity of the IL-8 receptor type CXCR1 and CXCR2 on human mast cells

To further elucidate mechanisms involved in mast cell accumulation at sites of cutaneous inflammation, we have studied the ability of human leukemic mast cells (HMC-1 cells) to express functionally active IL-8 receptors. Expression of mRNA for both types of IL-8 receptors (CXCR1 and CXCR2) was demonstrated by PCR and of both proteins by flow cytometry. Binding and competition studies with 125I-labeled IL-8 and its homologue melanoma growth stimulating activity (125I-labeled MGSA) revealed two specific binding sites for IL-8, K1 = 1.1 x 10(11) M(-1) and K2 = 5 x 10(7) M(-1); and for MGSA, K1 = 2.8 x 10(10) M(-1) and K2 = 5 x 10(7) M(-1). This finding was supported by a dose-dependent rise of cytosolic free calcium concentration ([Ca2+]i) induced by both chemokines and to a lesser extent by the homologue neutrophil-activating peptide-2 (NAP-2). A significant migratory response of human leukemic mast cells (HMC-1) was observed with all three chemokines at a range from 10(-8) M to 10(-9) M. Moreover, the formation of cellular F-actin was induced in a rapid, dose-dependent fashion, with a maximally 1.7-fold increase at 10(-7) M. Using postembedding immunoelectron microscopy, we could show the expression of CXCRI on the cytoplasmatic membrane of isolated human skin mast cells whereas CXCR2 was located in mast cell-specific granules. These findings demonstrate for the first time the functional expression of both types of IL-8 receptors on human mast cells, suggesting a role for their ligands during mast cell activation and recruitment.     

Isolation and functional characterization of IL-2 responsive T cell clones from NZB x NZW F1 mice

Autoantigen-reactive T cells might play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Autoantigen-reactive T cell clones were generated from spleens of NZB x NZW F1 (BWF1) and normal control BALB/c mice with interleukin-2 (IL-2), a procedure that selects for in vivo activated antigen-reactive T cells. The antigen-specificity of the T cell clones was tested by using a panel of candidate autoantigens. The T cell clones from BWF1 mice but not those from BALB/c mice proliferated against heparan sulfate, the major glycosaminoglycan of glomerular basement membrane. None of the clones proliferated against dsDNA or cardiolipin. All the heparan sulfate-reactive T cell clones had the ability to selectively augment the production of IgG anti-dsDNA autoantibodies. When cultured with either heparan sulfate or Concanavalin A, the T cell clones produced high levels of IL-4 and IL-5 with no detectable IL-2 or IFN-gamma. In contrast, T cell clones derived from BALB/c mice augmented the production of total polyclonal IgG but not the production of anti-dsDNA antibodies. These studies indicate the existence of heparan sulfate-reactive T cells in BWF1 mice. Characterization of heparan sulfate-reactive T cells that could selectively augment anti-dsDNA production will permit the design of targeted and antigen-specific therapy.     

Antibodies to domains II and III of the IL-1 receptor accessory protein inhibit IL-1 beta activity but not binding: regulation of IL-1 responses is via type I receptor, not the accessory protein

The IL-1R accessory protein (IL-1RAcP) plays a role in IL-1R signaling by forming a complex with IL-1RI bound to the IL-1 ligand. We identified four hydrophilic peptide regions of the extracellular IL-1RAcP that may be available for complex formation (peptide 1, 71-83 domain I; peptide 2, 204-211 domain II; peptide 3, 282-292 domain III; and peptide 4, 304-314 domain III). These peptides were synthesized, coupled to keyhole limpet hemocyanin, and used to produce rabbit antisera. Each affinity-purified antiserum showed specificity for the respective peptide without cross-reactivity. Anti-peptide 2, 3, and 4 recognized surface expression of IL-1RAcP on the Th2 D10S cells by FACS and inhibited IL-1-driven proliferation. Anti-peptide 4 recognized intact IL-1RAcP and soluble IL-1RAcP. Anti-IL-1RAcP-peptide 4, which targets the terminal segment of domain III, inhibited 80% of IL-1 beta-driven proliferation of D10S cells. However, these IL-1RAcP Abs had no effect on the activity of human or mouse IL-1 alpha. Whereas IL-1 beta down-regulated IL-1RI surface expression (p < 0.05), there was no change in the surface expression of IL-1RAcP. Moreover, murine IL-10 increased surface expression of IL-1RI, but did not affect expression of IL-1RAcP or the proliferation of D10S cells. Steady state levels of mRNA for IL-1RAcP and IL-1RI in D10S cells showed a similar pattern to that of surface expression of the respective receptors. We conclude that 1) blocking IL-1RAcP inhibits IL-1 signaling in D10S cells, 2) domains-II and III may be involved in complex formation with IL-1RI, 3) IL-1RAcP is not regulated as is IL-1RI in the same cells, and 4) IL-1 responsiveness is dependent on the expression of IL-1RI, not IL-1RAcP.     

Reconstitution of a functional interleukin (IL)-7 receptor demonstrates that the IL-2 receptor gamma chain is required for IL-7 signal transduction

The interleukin (IL)-2 receptor gamma chain has recently been shown to be a component of the IL-7 and IL-4 receptors. Using a transient transfection assay and the trans-activation of reporter gene constructs which are under the control of cytokine-responsive promoter elements, we have studied signal transduction through the IL-7 receptor (IL-7R). The reporter gene expression was not stimulated by receptors that contained the cytoplasmic domain of the IL-7R, either as intact IL-7R or as part of a chimeric receptor. However, co-expression of the IL-7R with the IL-2 receptor gamma chain was able to stimulate gene activation. For maximal stimulation the intact cytoplasmic domains of each chain was required.     

Knockout and reconstitution of a functional human type I interferon receptor complex

The functional subunits of the human Type I interferon (IFN) receptor complex have not been defined. Using site-specific recombination in a yeast artificial chromosome (YAC), we have produced a deletion within the human IFN-alpha receptor (Hu-IFN-alpha R1) gene which eliminates exon II of the gene. This deletion effectively eliminates the MHC Class I antigen induction and antiviral activity previously reported for this fully functional parental YAC clone (Soh, J., Mariano, T. M., Lim, J.-K., Izotova, L., Mirochnitchenko, O., Schwartz, B., Langer, J., and Pestka, S. (1994c) J. Biol. Chem. 269, 18102-18110). We have successfully reconstituted this activity by expression of the cDNA encoding the Hu-IFN-alpha R1 component (Uzé, G., Lutfalla, G., and Gresser, I. (1990) Cell 60, 225-234) in cells containing the YAC with this deletion. The Hu-IFN-alpha R1 subunit thus plays a critical role in the functional human Type I IFN receptor complex, whose components are encoded on this YAC. In addition, as binding of ligands is retained in the cells containing the YAC with the deletion, it is clear a second subunit encoded on the YAC is responsible for ligand binding activity. This system will now allow the identification of additional subunits involved in the response to the Type I IFNs and the functional significance of each.     

Interleukin 1 (IL-1) causes changes in lateral and rotational mobilities of IL-1 type I receptors

To investigate IL-1-dependent interactions of IL-1 type I (IL-1 RI) receptors on intact cells, lateral and rotational mobilities and detergent insolubility were investigated. Lateral mobility was measured by fluorescence photobleaching recovery, using a Cy3-modified, noncompetitive mAb specific for IL-1RI (M5) bound to wild-type IL-1 RI or mutant IL-1 RI with a truncated cytoplasmic tail. Addition of IL-1 causes significant reduction in the mobile fraction of wild-type IL-1 RI for two different transfected cell lines. For the mutant IL-1 RI, no significant decrease in response to IL-1 is observed, indicating that the missing cytoplasmic segment is involved in IL-1-dependent interactions of IL-1 RI that lead to reduced lateral mobility on the cell surface. The rotational mobility of IL-1 RI was assessed with phosphorescence anisotropy decay measurements using erythrosin-labeled M5. IL-1 decreases the rotational mobility of cell surface IL-1 RI on the microsecond time scale and also increases the initial anisotropy, indicating loss in segmental motion. Measurements of resistance to solubilization by Triton X-100 showed that IL-1 binding increases the fraction of IL-1 RI sedimenting with cytoskeletal residues. The IL-1 receptor antagonist protein (IL-1ra) causes partial effects in reducing rotational mobility and increasing detergent insolubility of M5-lableled IL-1 RI, indicating that this ligand causes structural changes in the presence of the dimerizing M5 mAb. These ligand-dependent physical interactions of IL-1 RI on the cell surface may be related to signal initiation by this receptor.     

Enhanced Th2-like responses in IL-1 type 1 receptor-deficient mice

IL-1 has a number of effects on T cell growth but a specific role for IL-1 in T cell responses in vivo has not been elucidated. In this study the role of IL-1 in Th1/Th2 responses was examined in mice deficient for the IL-1 type 1 receptor (IL-1RI-/-) during cutaneous Leishmania major infection or following immunization with keyhole limpet hemocyanin (KLH). After inoculation of L. major stationary phase promastigotes into the hind footpad, both IL-1RI-/- and wild-type (WT) mice developed small lesions which resolved spontaneously. Lymph node cells from infected IL-1RI-/- mice produced significantly more IL-4 and IL-10 than those from WT mice following antigenic stimulation in vitro. Splenocytes from IL-1RI-/- and WT mice showed similar levels of antigen-induced proliferation. In contrast, splenocyte cultures from the IL-1RI-/- mice contained significantly more IL-4 than those from WT mice. Similar results were also obtained after immunization with KLH. While lymph node cells from both IL-1RI-/- and WT mice displayed similar levels of KLH-specific proliferation, those from IL-1RI-/- mice produced significantly more IL-4 than those from WT mice. Conversely, antigen-stimulated lymph node cells from WT mice secreted significantly greater amounts of IFN-gamma as compared with those from IL-1RI-/- mice. These data indicate that while IL-1 is not required for mounting an immune response or antigen-dependent proliferation, it appears to be required for normal regulation of Th1/Th2 responses and may function to negatively regulate IL-4 expression.     

Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor

We used KO mice lacking either TNF receptor 1 (TNFR-1) or receptor 2 (TNFR-2) to determine whether signaling at the start of liver regeneration after partial hepatectomy (PH) involves only one or both TNF receptors and to analyze in more detail the abnormalities caused by lack of TNFR-1 receptor, which is required for the initiation of liver regeneration. Lack of TNFR-2 had little effect on NF-kappaB and STAT3 binding, and no effect in interleukin-6 production after PH, but caused a delay in AP-1 and C/EBP binding and in the expression of c-jun and c-myc messenger RNA (mRNA). In contrast to mice lacking TNFR-1, which had deficient hepatocyte DNA synthesis and massive lipid accumulation in hepatocytes, TNFR-2 KO mice had normal liver structure and similar levels of hepatocyte DNA replication as those of wild type mice. We conclude that TNFR-1, but not TNFR-2, is necessary for liver regeneration, and that NF-kappaB and STAT3 binding are activated by signals transduced by TNFR-1. Inhibition of AP-1 and C/EBP binding and in the expression of c-jun and c-myc mRNA in the first 4 hours after PH, as well as the apparent lack of Fos in AP-1 complexes, had no effect on the timing or extent of DNA replication.     

Impaired IL-1beta-induced neutrophil accumulation in tachykinin NK1 receptor knockout mice

Tachykinin NK1 receptors play an important role in the development of neurogenic inflammatory responses. We have used the murine air-pouch model to investigate whether the neurogenic component of the cellular inflammatory response to interleukin-1beta (IL-1beta, 10 ng into the air-pouch) is altered in NK1 receptor knockout mice compared to wild type controls. Air-pouches were washed following a 4 h IL-1beta treatment, the wash collected and neutrophil number estimated using a Neubauer haemocytometer. The response to IL-1beta was significantly attenuated in NK1 receptor +/- (40% reduction) and -/- mice (62% reduction) compared to wild type controls (+/+), whilst the response to cytokine-induced neutrophil chemoattractant (CINC, 0.3 microg) was unaffected. The response to substance P (7.5 nmol) was attenuated by approximately 50% in both NK1 receptor +/- and -/- mice compared to wild type controls. In conclusion NK1 receptors play a significant role in the cellular response to IL-1beta in a model of inflammation.     

The murine IL-13 receptor alpha 2: molecular cloning, characterization, and comparison with murine IL-13 receptor alpha 1

Two components of a receptor complex for IL-13, the IL-4R and a low affinity IL-13-binding chain, IL-13R alpha 1, have been cloned in mice and humans. An additional high affinity binding chain for IL-13, IL-13R alpha 2, has been described in humans. We isolated a cDNA from the thymus that encodes the murine orthologue of the human IL-13R alpha 2. The predicted protein sequence of murine IL-13R alpha 2 (mIL-13R alpha 2) has 59% overall identity to human IL-13R alpha 2 and is closely related to the murine low affinity IL-13-binding subunit, IL-13R alpha 1. The genes for both mIL-13-binding chains map to the X chromosome. A specific interaction between mIL-13R alpha 2.Fc protein and IL-13 was demonstrated by surface plasmon resonance using a BIACORE instrument. Ba/F3 cells that were transfected with mIL-13R alpha 2 expressed 5000 molecules per cell and bound IL-13 with a single Kd of 0.5 to 1.2 nM. However, these cells did not proliferate in response to IL-13, and the IL-4 dose response was unaffected by high concentrations of IL-13. In contrast, the expression of mIL-13R alpha 1 by Ba/F3 cells resulted in a sensitive proliferative response to IL-13. Consistent with its lower affinity for IL-13, IL-13R alpha 1.Fc was 100-fold less effective than IL-13R alpha 2.Fc in neutralizing IL-13 in vitro. These results show that mIL-13R alpha 2 and mIL-13R alpha 1 are not functionally equivalent and predict distinct roles for each polypeptide in IL-13R complex formation and in the modulation of IL-13 signal transduction.     

Phenotypic characterization of a patient homozygous for the D558N LDL receptor gene mutation

We describe the clinical, biochemical, and genetic features of a patient with true homozygous familial hypercholesterolemia due to the D558N low-density lipoprotein receptor gene mutation, previously designated FH Cincinnati-4. Functional flow-cytometric analysis of the LDL receptorR protein on upregulated EBV-transformed lymphocytes indicated reduction of the number of receptors on the cell surface by 87% and reduction of receptor activity by 89% compared to control cells. With drugs and a portacaval shunt operation, performed when the patient was 15 years old, serum cholesterol was reduced from about 28 to about 15 mmol/l. He died at the age of 32 of a myocardial infarction. The autopsy showed generalized atherosclerosis, especially in the coronary arteries, which were severely stenosed proximally. A rare finding was a large intracranial xanthoma that apparently had been asymptomatic.     

Mechanical deformation promotes secretion of IL-1 alpha and IL-1 receptor antagonist

Both IL-1 alpha and IL-1 beta lack an N terminus secretory sequence, and the mechanism of secretion of these pleiotropic cytokines is incompletely understood. The epidermis contains large quantities of IL-1 alpha in keratinocytes, which may play a role in inducing endothelial adhesion molecules and promoting extravasation of leukocytes. Here we report that mechanical deformation of human keratinocytes leads to rapid release of IL-1 alpha, possibly through transient disruptions in the plasma membrane. Using a device that precisely controls the amplitude of strain on the culture substrate, we found by pulse-chase analysis, Western analysis, and ELISA that the release of IL-1 alpha is dependent on the amplitude of the strain. A cyclic strain of 14% released a small but significant quantity of IL-1 alpha, while strains of 33% released 66 +/- 9% of cytoplasmic IL-1 alpha over 1 h (p < 0.001). Release of IL-1 alpha was accompanied by rapid release of large stores of IL-1R antagonist, approximately 25 to 30 times greater by mass than the quantity of IL-1 alpha released, but only a small fraction of cytoplasmic lactate dehydrogenase. Media conditioned by mechanically stimulated keratinocytes induced expression of E-selectin by human vascular endothelial cells; induction of E-selectin was completely inhibited by an Ab to IL-1 alpha. Therefore, mechanical strain promotes the secretion of IL-1 alpha, and deformation of keratinocytes in the epidermis may activate vascular endothelium through mechanically released IL-1 alpha. This pathophysiologic mechanism may play a role in the anatomic localization of some inflammatory skin diseases, such as psoriasis, which occurs more commonly in locations where the dermis is subjected to repetitive stretch or trauma.     

Expression of type II nitric oxide synthase in primary human astrocytes and microglia: role of IL-1beta and IL-1 receptor antagonist

In this work, we studied the expression of type II nitric oxide synthase (NOS) in primary cultures of human astrocytes and microglia. Cytokine-activated human fetal astrocytes expressed a 4.5-kb type II NOS mRNA that was first evident at 8 h, steadily increased through 48 h, and persisted through 72 h. The inducing signals for astrocyte NOS II mRNA expression were in the order IL-1beta + IFN-gamma > IL-1beta + TNF-alpha > IL-1beta. SDS-PAGE analysis of cytokine-stimulated astrocyte cultures revealed an approximately 130-kDa single NOS II band that was expressed strongly at 48 and 72 h (72 h > 48 h). Specific NOS II immunoreactivity was detected in cytokine-treated astrocytes, both in the cytosol and in a discrete paranuclear region, which corresponded to Golgi-like membranes on immunoelectron microscopy. In human microglia, cytokines and LPS failed to induce NOS II expression, while the same stimuli readily induced TNF-alpha expression. In cytokine-treated human astrocytes, neither NOS II mRNA/protein expression nor nitrite production was inhibited by TGF-beta, IL-4, or IL-10. In contrast, IL-1 receptor antagonist exerted near complete inhibition of NOS II mRNA and nitrite induction. Monocyte chemoattractant peptide-1 mRNA was induced in TGF-beta-treated astrocytes, demonstrating the presence of receptors for TGF-beta in astrocytes. These results confirm that in humans, cytokines stimulate astrocytes, but not microglia, to express NOS II belonging to the high output nitric oxide system similar to that found in rodent macrophages. They also show that the regulation of type II NOS expression in human glia differs significantly from that in rodent glia. A crucial role for the IL-1 pathway in the regulation of human astrocyte NOS II is shown, suggesting a potential role for IL-1 as a regulator of astrocyte activation in vivo.