Susan M. MacDonald

Immune mimicry in malaria: Plasmodium falciparum secretes a functional histamine-releasing factor homolog in vitro and in vivo

The Plasmodium falciparum translationally controlled tumor protein (TCTP) is a homolog of the mammalian histamine-releasing factor (HRF), which causes histamine release from human basophils and IL-8 secretion from eosinophils. Histamine, IL-8, and eosinophils have been reported to be elevated in patients with malaria. This study was undertaken to determine whether malarial TCTP is found in the plasma of malaria-infected patients and to determine whether it has HRF biologic activity. Malarial TCTP was found in lightly infected human volunteers and in heavily infected Malawian children, but not in uninfected patients. Recombinant malarial TCTP, like HRF, stimulated histamine release from basophils and IL-8 secretion from eosinophils in vitro. Whereas malarial TCTP was less active than HRF, the concentrations that were effective in vitro could be achievable in vivo. These data suggest that malarial TCTP, present in human plasma during a malarial illness, may affect host immune responses in vivo.

The human recombinant histamine releasing factor: Functional evidence that it does not bind to the IgE molecule☆☆☆★

BACKGROUND We have previously shown that the human recombinant histamine releasing factor (HrHRF) caused histamine release from a subset of basophils from donors with allergy, and this release seemed to be dependent on the presence of a certain type of IgE, termed IgE+. IgE molecules that did not support HrHRF-induced histamine release were termed IgE-. However, subsequently we demonstrated that HrHRF primes anti-IgE-antibody-induced histamine release from all basophils, irrespective of the type of IgE on the cell surface. OBJECTIVE Because these data suggested that HrHRF does not exert its biologic effects by binding to IgE, but rather that it interacted with a surface receptor on the basophil, we wanted to obtain functional evidence that HrHRF did or did not bind to the IgE molecule. METHODS The rat basophilic leukemia cell line (RBL-SX38), which has been transfected to express a functional human FcepsilonRI (alpha-, beta-, and gamma-chains of the receptor) in addition to the normal rat FcepsilonRI, was used. The presence of the human FcepsilonRI receptor enables these cells to be sensitized with human IgE. Cells were passively sensitized with 1000 ng/mL human IgE+ or 1000 ng/mL human IgE- for 60 minutes at 37 degrees C. Unsensitized cells served as a control. After the cells were washed, 1 x l0(5) cells were stimulated in the presence of 1 mmol/L Ca2+ with 0.1 microg/mL anti-IgE, 40 microg/mL HrHRF, or 40 microg/mL mouse recombinant HRF (MrHRF), which has 96% homology to HrHRF. RESULTS Mean anti-IgE-induced histamine release was 33% +/- 15%, and there was no difference between IgE+ sensitization (32% +/- 12%) and IgE- sensitization (34% +/- 18%). However, in contrast to human basophil experiments, neither HrHRF (0% +/- 0%) nor MrHRF (3% +/- 5%) caused histamine release in RBL cells sensitized with IgE+. In addition, priming the transfected RBL-SX38 cells or the parental cell line, RBL-2H3 cells, with HrHRF or MrHRF did not increase anti-IgE-induced histamine release. CONCLUSION The results indicate that HrHRF does not bind to IgE, either IgE+ or IgE-. Therefore it appears likely that rHRF signals through its own specific receptor, which is not expressed or functional on RBL-SX38 or RBL-2H3 cells, but which seems to be expressed on basophils of atopic and nonatopic donors.

Histamine-releasing factors

There is increasing evidence that human basophils accumulate at sites of chronic inflammation, and, in particular, in allergic asthma. Investigators have, therefore, become very interested in identifying proteins that activate these cells. Recently, the gene encoding a candidate for this function, a novel molecule, the IgE-dependent histamine-releasing factor, was cloned.

Selectivity of Mediators Released by Eosinophils

The presence of receptors for IgE on eosinophils has drawn the attention on their direct participation in IgE-dependent hypersensitivity reactions. Surface IgE antibodies were detected on eosinophils from allergic patients. The addition of the specific allergen or anti-IgE antibodies to such purified eosinophils induced the release of eosinophil peroxidase, but not of eosinophil cationic protein. These findings associated with results obtained by using electron microscopy and immunogold staining of the various antibodies directed against the granule proteins allowed us to suggest a selectivity in the mediators released by eosinophils. In addition, preliminary results concerning the existence and the functional role of a receptor for IgA on eosinophils are reported, leading to the concept of a particular interaction of eosinophils with immunoglobulins present in the tissues and their participation in local immune responses.

Histamine-releasing factor/translationally controlled tumor protein (HRF/TCTP)-induced histamine release is enhanced with SHIP-1 knockdown in cultured human mast cell and basophil models

Previously, we demonstrated a negative correlation between histamine release to histamine‐releasing factor/translationally controlled tumor protein (HRF/TCTP) and protein levels of SHIP‐1 in human basophils. The present study was conducted to investigate whether suppressing SHIP‐1 using small interfering (si)RNA technology would alter the releasability of culture‐derived mast cells and basophils, as determined by HRF/TCTP histamine release. Frozen CD34+ cells were obtained from the Fred Hutchinson Cancer Research Center (Seattle, WA, USA). Cells were grown in StemPro‐34 medium containing cytokines: mast cells with IL‐6 and stem cell factor (100 ng/ml each) for 6–8 weeks and basophils with IL‐3 (6.7 ng/ml) for 2–3 weeks. siRNA transfections were performed during Week 6 for mast cells and Week 2 for basophils with siRNA for SHIP‐1 or a negative control siRNA. Changes in SHIP‐1 expression were determined by Western blot. The functional knockdown was measured by HRF/TCTP‐induced histamine release. siRNA knockdown of SHIP‐1 in mast cells ranged from 31% to 82%, mean 65 ± 12%, compared with control (n=4). Histamine release to HRF/TCTP was increased only slightly in two experiments. SHIP‐1 knockdown in basophils ranged from 34% to 69%, mean 51.8 ± 7% (n=4). Histamine release to HRF/TCTP in these basophils was dependent on the amount of SHIP knockdown. Mast cells and basophils derived from CD34+ precursor cells represent suitable models for transfection studies. Reducing SHIP‐1 protein in cultured mast cells and in cultured basophils increases releasability of the cells.

Pharmacologic regulation of histamine release by the human recombinant histamine-releasing factor

BACKGROUND The recently cloned human recombinant IgE-dependent histamine releasing factor (HrHRF) was initially thought to stimulate histamine release from human basophils from a subpopulation of allergic donors by interacting with the IgE molecules on the surface of these cells. Additional data suggest that HrHRF exerts its biologic effects by binding to a distinct cell surface structure and not to IgE. OBJECTIVE To address the hypothesis that the HrHRF signaling pathway is distinct from the classical high-affinity IgE receptor (FcepsilonRI) pathway, we used pharmacologic agents known to affect basophil histamine release. METHODS In this report we compared the effect of staurosporine, Bis II, Gö 6976, rottlerin, and pertussis toxin on histamine release from human basophils mediated by the following stimuli: HrHRF, polyclonal human anti-IgE antibody, and antigen, as well as the IgE-independent stimulus, FMLP. RESULTS None of these modulators, except rottlerin, could differentiate histamine release induced by anti-IgE or antigen from that induced by HrHRF. Rottlerin enhanced HrHRF-mediated histamine release and dose dependently blocked FMLP-mediated release without affecting basophil activation by either anti-IgE or antigen. CONCLUSION These data suggest a unique signaling pathway for HrHRF and thus strengthen the hypothesis that HrHRF binds to a specific receptor other than IgE.

H2O2-dependent translocation of TCTP into the nucleus enables its interaction with VDR in human keratinocytes: TCTP as a further module in calcitriol signalling

Translationally controlled tumour protein (TCTP) is an evolutionarily highly conserved molecule implicated in many processes related to cell cycle progression, proliferation and growth, to the protection against harmful conditions including apoptosis and to the human allergic response. We are showing here that after application of mild oxidative stress, human TCTP relocates from the cytoplasm to the nuclei of HaCaT keratinocytes where it directly associates with the ligand-binding domain of endogenous vitamin D(3) receptor (VDR) through its helical domain 2 (AA 71-132). Interestingly, the latter harbours a putative nuclear hormone receptor coregulatory LxxLL-like motif which seems to be involved in the interaction. Moreover, we demonstrate that VDR transcriptionally induces the expression of TCTP by binding to a previously unknown VDR response element within the TCTP promotor. Conversely, ectopically overexpressed TCTP downregulates the amount of VDR on both mRNA as well as protein level. These data, to conclude, suggest a kind of feedback regulation between TCTP and VDR to regulate a variety of (Ca(2+) dependent) cellular effects and in this way further underscore the physiological relevance of this novel protein-protein interaction.

Induced Loss of Syk in Human Basophils by Non-IgE-Dependent Stimuli

In the general population, Syk expression in human basophils is highly variable and correlates well with the IgE-mediated responsiveness of these cells. Previous studies established that IgE-mediated stimulation results in loss of Syk expression. The current studies investigated whether stimulation through other receptors results in loss of Syk. Two classes of stimulation were examined, those that operate through the kinase Syk and those that operate through a GTP-binding protein. These studies demonstrated that aggregation of leukocyte Ig-like receptor LILRA-2 resulted in phosphorylation of Syk and c-Cbl, was inhibited by a third generation Syk inhibitor with an expected IC50, and induced histamine release in strict proportion to release induced by anti-IgE Ab. Stimulation of LILRA-2 for 18 h resulted in modest loss of Syk that correlated with the more profound loss of Syk induced by anti-IgE Ab. Human recombinant histamine-releasing factor has also recently been shown to induce Syk phosphorylation and in the current studies has also been shown to induce loss of Syk in 18-h cultures. fMLP stimulation for 18 h was also found to induce modest loss of Syk. fMLP induced phosphorylation of c-Cbl that was sustained for at least 45 min. Phosphorylation of c-Cbl was inhibited by a Syk kinase inhibitor but with an IC50 that was not consistent with Syk activity, suggesting another kinase was responsible for Cbl phosphorylation following fMLP. These studies demonstrate that it is possible to induce the loss of Syk expression in human basophils by a non-IgE-dependent mechanism and even by a mechanism that does directly involve Syk in the reaction complex.

Inhibition of Cytokine Gene Transcription by the Human Recombinant Histamine-Releasing Factor in Human T Lymphocytes

Human recombinant histamine-releasing factor (HrHRF) preincubation enhances the secretion of histamine, IL-4, and IL-13 from FcεRI-stimulated human basophils. In GM-CSF-primed human eosinophils, HrHRF increases IL-8 production. Our recent experiments were designed to evaluate the effects of HrHRF on human T cell cytokine production. Purified T cells were preincubated with GST-tagged HrHRF, followed by stimulation with PMA and A23187 overnight. A partial inhibition of IL-2 and IL-13 production (30 and 75%, respectively) was detected compared with that in cells treated with PMA/A23187 alone. However, the production of IFN-γ was similar in PMA/A23187 stimulated cells with or without HrHRF. The inhibition of cytokine protein production was dose dependent and specific to the HrHRF portion of GST-HrHRF. The inhibition was not due to endotoxin, since preincubation with polymyxin B and HrHRF gave similar results to that with HrHRF alone. The same pattern and specificity of cytokine regulation were replicated in the Jurkat T cell line as for primary T cells. The PMA/A23187-stimulated activity of a proximal promoter IL-13, IL-4, or IL-2 luciferase construct transfected into Jurkat cells was partially inhibited (60, 32, or 70%, respectively) upon GST-HrHRF preincubation, suggesting that HrHRF functions to inhibit cytokine production in Jurkat cells by preventing gene transcription. The inhibition of IL-2 promoter activation was specific to the HrHRF portion of GST-HrHRF. We conclude that HrHRF, in addition to functioning as a histamine-releasing factor, can differentially modulate the secretion of cytokines from human basophils, eosinophils, T cells, and murine B cells, suggesting that it may induce a complex array of responses at sites of allergic inflammation.

Human Recombinant Histamine-Releasing Factor

Histamine-releasing factors (HRF) have received much attention over the last 17 years. With the availability of molecular biology techniques, cloned molecules have been identified as HRF. Examples include various interleukins, chemokines and the IgE-dependent HRF This human recombinant HRF (HrHRF) is a novel cytokine with no homology to any known molecule. Indeed, HrHRF acts as a complete secretagogue for basophils possessing a certain type of IgE, namely IgE+. Additionally, HrHRF can enhance histamine release from basophils possessing IgE-, basophils not normally responsive to HrHRF. Furthermore, HrHRF can activate eosinophils from allergic donors. This unique molecule may have a pivotal role in allergic diseases.