LAD2 Mast Cells Research Articles

Advances in mechanisms of asthma, allergy, and immunology in 2008

This review summarizes selected articles appearing in 2008 in the Journal. Articles chosen include those improving our understanding of mechanisms of allergic diseases by focusing on human basophil, mast cell, and eosinophil biology; IgE and its high-affinity receptor on various cells; novel properties of omalizumab; airways remodeling; and genetics. Articles from other journals have been included to supplement the topics presented.

Substance P Modulates Toll-like Receptor-Mediated Activation Of Human Mast Cells

RATIONALE: Substance P (SP) is a neuropeptide with potent neuroimmunoregulatory activity that may play a role in susceptibility to infection. We examined human mast cell responses to SP and toll-like receptors (TLR) ligands, pathogen-associated molecules that initiate innate immunity. METHODS: Human cultured mast cells (LAD2) were stimulated with SP or IgE/anti-IgE and expression of TLR was assessed by real-time PCR. LAD2 mast cells were activated by SP and TLR ligands including lipopolysaccharide (LPS; 100 ng/mL), Pam3CSK4 (10 ug/mL) and lipoteichoic acid (LTA; 10 ug/mL) and mast cell leukotriene and chemokine production was assessed by ELISA. Mast cell degranulation was determined by beta-hexosaminidase (beta-hex) assay. RESULTS: SP treatment of LAD2 upregulated mRNA for TLR2, 4 and 9 (3.4, 4.4 and 2.9 fold compared to untreated respectively, p < 0.01) while anti-IgE stimulation upregulated expression of TLR4 only (3.4 fold compared to untreated p < 0.01). Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA potentiated production of LTC4 compared to treatment with Pam3CSK4 or LTA alone (2.6 and 2.1 fold over control respectively p < 0.01). Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. Pretreatment with Pam3CSK followed by SP activation attenuated LAD2 degranulation compared to SP alone (36.4 + 1.3 vs. 44.0 + 2.9% beta-hex release, p < 0.01). Pretreatment with LTA or LPS followed by SP stimulation synergistically induced expression of CXCL8/IL-8 mRNA and production of CXCL8/IL-8 and CCL2/MCP-1 protein. CONCLUSIONS: We demonstrate that SP and TLR ligands regulate mast cell activation. These results suggest that neuronal responses may influence host defense responses and vice versa.

CD27-CD70 interactions in the pathogenesis of Waldenström macroglobulinemia

AbstractWaldenström macroglobulinemia (WM) is a B-cell malignancy characterized by an IgM monoclonal gammopathy and bone marrow (BM) infiltration with lymphoplasmacytic cells (LPCs). Excess mast cells (MCs) are commonly present in WM, and provide growth and survival signals to LPCs through several TNF family ligands (CD40L, a proliferation-inducing ligand [APRIL], and B-lymphocyte stimulator factor [BLYS]). As part of these studies, we demonstrated that WM LPCs secrete soluble CD27 (sCD27), which is elevated in patients with WM (P < .001 vs healthy donors), and serves as a faithful marker of disease. Importantly, sCD27 stimulated expression of CD40L on 10 of 10 BM MC samples and APRIL on 4 of 10 BM MC samples obtained from patients with WM as well as on LAD2 MCs. Moreover, the SGN-70 humanized monoclonal antibody, which binds to CD70 (the receptor-ligand partner of CD27), abrogated sCD27 mediated up-regulation of CD40L and APRIL on WM MCs. Last, treatment of severe combined immunodeficiency–human (SCID-hu) mice with established WM using the SGN-70 antibody blocked disease progression in 12 of 12 mice, whereas disease progressed in all 5 untreated mice. The results of these studies demonstrate a functional role for sCD27 in WM pathogenesis, along with its utility as a surrogate marker of disease and a target in the treatment of WM.

Mast cells and fibroblast-like synoviocytes co-culture increases interleukin-6 secretion in rheumatoid arthritis

To explore the significance of mast cells-fibroblast like synoviocytes (FLS) interaction in the pathogenesis of rheumatoid arthritis (RA). Frozen sections of RA synovia from 4 patients were made to observe the distribution of mast cells by immunochemistry. FLSs were isolated from 4 samples of RA synovia and co-cultured with the human mast cells of the line LAD-2 for 0, 12, 24, and 48 h, and 7 d. ELISA was used to detect the interleukin (IL)-6, an important proinflammatory cytokine. 4% formaldehyde was used to fix the FLS and LAD-2 cells and then detect the IL-6 levels in the supernatant. Other FLS and LAD-2 mast cells were put into the upper and lower chambers of Transwell system to be cocultured in isolate condition, an important proinflammatory cytokine. Microscopy showed that FLSs were adjacent to the mast cells. The IL-6 content of the FLS/LAD-2 cell co-culture supernatant was 7150 microg/L +/- 114 microg/L, significantly higher than that of the FLS supernatant (3019 microg/L +/- 57 microg/L, P < 0.01). LAD-2 cell number dose-dependently increased the IL-6 content in the supernatant. Almost no IL-6 secretion was found in the supernatant of the co-culture with fixed FLSs and non-fixe4d LAD-2 cells, and the IL-6 secretion in the supernatant of the co-culture with fixed LAD-2 cells and non-fixed FLSs was similar to that of the single FLS group and far lower than that of the FLS-LAD-2 co-culture group. Transwell system test showed that there was no significant difference in the IL-5 level between the direct mixture co-culture group and indirect mixture co-culture. Mast cell/FLS interaction increases the IL-6 secretion via soluble mediators, thus playing an important role in the pathogenesis of RA.

Preclinical In Vitro and In Vivo Evidence Support a Therapeutic Role for the CD70 Directed Monoclonal Antibody (SGN-70) in Waldenström's Macroglobulinemia (WM).

Introduction: WM is a B-cell disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells (LPC), along with excess mast cells (MC) which support the growth and survival of BM LPC through multiple TNF-family ligands including CD40L, APRIL and BLyS/BAFF. Importantly, BM LPC stimulate cell surface expression of TNF-family ligands through release of sCD27 which induces CD70 on MC. We therefore have sought the development of agents which could target CD27-CD70 interactions. As such, we examined the therapeutic potential of directly targeting CD70 using the fully humanized monoclonal antibody SGN-70 (Seattle Genetics, Inc., Bothell WA).Methods-Results: As part of these studies, we used flow cytometric analysis to evaluate the expression of CD70 on primary WM patient BM LPC and MC, as well as 2 WM cell lines (BCWM.1 and WM-WSU). These studies demonstrated cell surface expression of CD70 on BM LPC and MC from 20/26 (77%) and 10/11 (90%) WM patients, respectively. We next assessed the ability of the SGN-70 antibody to eradicate primary WM LPC (n=5) and WM cell lines by assessing for direct induction of apoptosis, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) as well as induction of TNF family ligands on primary WM MC and the LAD2 MC line. Following incubation of WM LPC with SGN-70 (0.01–20 μg/ml), no direct induction of apoptosis or CDC activity was observed. However, SGN-70 mediated significant dose-dependent ADCC against WM LPC and MC at concentrations of 0.1–20 ug/ml. Importantly, SGN-70 blocked sCD27-induced expression of CD40L and APRIL on primary WM MC and LAD2 MC. To further evaluate the therapeutic potential of SGN-70 in an in vivo model, SCID-hu mice bearing BCWM.1 WM cells were treated with SGN-70 (1 mg/kg, i.p., qOD) Serum human IgM and sCD27 levels were measured by ELISA to monitor for tumor engraftment and disease progression. SGN-70 initiated 6 weeks following tumor engraftment blocked tumor growth in 12/12 treated mice, whereas all 5 untreated mice demonstrated disease progression. The results of these studies provide the framework for clinical trials to examine the therapeutic potential of the SGN-70 monoclonal antibody in WM.

Role of ABCC1 in export of sphingosine-1-phosphate from mast cells

Mast cells play a pivotal role in inflammatory and immediate-type allergic reactions by secreting a variety of potent inflammatory mediators, including sphingosine-1-phosphate (S1P). However, it is not known how S1P is released from cells. Here, we report that S1P is exported from mast cells independently of their degranulation and demonstrate that it is mediated by ATP binding cassette (ABC) transporters. Constitutive and antigen-stimulated S1P release was inhibited by MK571, an inhibitor of ABCC1 (MRP1), but not by inhibitors of ABCB1 (MDR-1, P-glycoprotein). Moreover, down-regulation of ABCC1 with small interfering RNA, which decreased its cell surface expression, markedly reduced S1P export from both rat RBL-2H3 and human LAD2 mast cells. Transport of S1P by ABCC1 influenced migration of mast cells toward antigen but not degranulation. These findings have important implications for S1P functions in mast cell-mediated immune responses.

Alteration and acquisition of Siglecs during in vitro maturation of CD34+ progenitors into human mast cells

Using human mast cells (MC) derived by culture of CD34+ peripheral blood precursors, a comprehensive study was performed of expression of 11 known Siglecs. Analysis was initially performed at the mRNA level using gene arrays. Positive results were then validated at the protein level using indirect immunofluorescence and flow cytometry, and for some Siglecs, Western blot analysis was also used. Culture-derived MC expressed mRNA for CD22 (Siglec-2), CD33 (Siglec-3), Siglec-5, Siglec-6, Siglec-8 and Siglec-10. Flow cytometry confirmed surface expression of all these molecules except for CD22 and Siglec-10, where levels were low or undetectable. However, Western blotting was able to detect MC expression of CD22 and Siglec-10, suggesting that these proteins were mostly cytoplasmic. CD34+ precursor cells from peripheral blood constitutively expressed surface CD33, Siglec-5 and Siglec-10. As they matured into MC, their constitutive levels of CD33 changed little, Siglec-5 and Siglec-10 declined, and Siglec-6 and Siglec-8 appeared de novo, all in parallel with accumulation of histamine and other MC markers, such as surface expression of FcepsilonRIalpha, and CD51. Phenotypic analysis of LAD-2 MC yielded a similar pattern of Siglec expression except that CD22 expression was particularly prominent. Finally, immunohistochemistry confirmed expression of these same Siglecs by mature tryptase-positive MC in human lung tissues. These data demonstrate an extensive and previously unappreciated pattern of Siglec expression on human MC. Whether engagement and signaling through these inhibitory Siglecs can impact MC biology will require further investigation.

CD52 Is Expressed on Human Mast Cells and Is a Potential Therapeutic Target in Waldenström's Macroglobulinemia and Mast Cell Disorders

BackgroundAlemtuzumab is a monoclonal antibody used in the treatment of CD52-expressing B-cell malignancies, including Waldenström's macroglobulinemia (WM). Recent studies demonstrate high levels of alemtuzumab activity in relapsed/refractory disease. One potential target of alemtuzumab is bone marrow mast cells (BMMCs), which provide growth and survival signaling for WM lymphoplasmacytic cells. Patients and MethodsWe therefore examined BMMCs (FcɛRI+, CD117+) from WM and other mast cell (MC) disorders for expression of CD52. ResultsWe identified cell surface antigen expression by multicolor flow cytometric analysis and found CD52 expressed on human mast-derived cell line–1 (HMC-1) and LAD2 MC lines, on BMMC from 13 of 15 patients with WM, and on BMMCs from 4 of 4 patients with systemic mastocytosis (SM). None of 4 healthy donors expressed CD52. Reverse-transcriptase polymerase chain reaction analysis confirmed CD52 expression in the HMC-1 and LAD2 MC lines, in BMMCs from 14 of 15 patients with WM, and 3 of 3 patients with SM. CD52 transcripts were also detected in BMMCs from 6 of 6 healthy donors, despite the absence of CD52 cell surface expression. Importantly, we observed high levels of alemtuzumab-mediated, antibody-dependent, cell-mediated cytotoxicity against LAD2 MCs and BMMCs from patients with WM and SM. ConclusionThese studies demonstrate that CD52 is widely expressed on human MCs and WM bone marrow lymphoplasmacytic cells and provide the preclinical rationale for the use of alemtuzumab in the treatment of WM and possibly other MC-related disorders.

Use of the LAD-2 Mast Cell Line to Assess Mast Cell Degranulation Induced by Sera From Chronic Idiopathic Urticaria Patients

Use of the LAD-2 Mast Cell Line to Assess Mast Cell Degranulation Induced by Sera From Chronic Idiopathic Urticaria Patients

Evidence for a restricted rather than generalized stimulatory response of skin-derived human mast cells to substance P

To resolve the controversy regarding substance P (SP) mediated stimulation of mast cells (MC), we demonstrate that SP triggers histamine release from purified human skin MC (sMC), but contrast to stimulation via FcɛRI, does not effect the production of TNF-α or IL-8. Conversely, both anti-IgE and SP are suppressive in terms of IL-6. By quantitative RT-PCR, the amount of templates at baseline (per 25 ng total RNA) is 2178 (IL-6), 2,665 (IL-8) and 94 (TNF-α), and remains unaltered by SP. Contrast to sMC, LAD2 MC respond to SP with stronger histamine release and robust TNF-α production in an only partially neurokinin-1R mediated manner, while histamine release of sMC is chiefly mediated by this receptor. We conclude that human sMC are responsive to SP in a selective manner by eliciting degranulation without the induction of cytokines and that SP-triggered cytokine production varies among MC subtypes, likely through differences in signaling mechanisms.

Differentiation, Distribution, and Chemical State of Intracellular Trace Elements in LAD2 Mast Cell Line

Oxidation state changes of metallic ions are involved in the generation and biological defense against reactive oxygen species. The relationship between allergy and oxidative damage by metallic elements was studied by X-ray fluorescence analysis using a mast cell line. The distribution of metallic elements is changed by the induction of reactive oxygen species. In mast cells, the degranulation leading to antigen or calcium ionophore stimulation is related to excessive accumulation of iron and to its chemical state. X-ray absorption near-edge structure spectroscopy showed that the oxidation state of iron in the cells shifted from Fe(II) to Fe(III) in degranulation. This finding might have implications for understanding the mechanisms involved in IgE-mediated cell responses as seen in allergic reaction.

Expression of CD33-related Siglecs on human eosinophils, basophils and mast cells

Background The Siglec family of cell surface molecules, of which Siglec-3 (CD33) is a member, shares properties including cytoplasmic ITIM motifs. One member, Siglec-8, has two isoforms, Siglec-8 and Siglec-8L, with the latter expressing ITIMs that could be responsible for its proapoptotic effects. Rationale Expression patterns of Siglec-3-related structures by eosinophils, basophils, and mast cells have not been compared. Objective We investigated baseline and activation-related expression of Siglecs −3, −5, −8 and −10 on these cells. Methods RT-PCR was used to detect mRNA expression. Specific antibodies recognizing Siglecs were used for flow cytometry or western blotting. For priming, eosinophils or basophils were cultured with or without cytokines for 18 hours. Results mRNA for the Siglec-8L isoform was detected in all eosinophil preparations (n=6); most but not all also expressed mRNA for Siglec-8. Basophils, HMC-1, LAD-2 and cord blood-derived mast cells consistently expressed both Siglec-8 isoforms. This was confirmed by western blotting, which revealed both 45 and 75 kDa proteins. Total surface Siglec-8 and −8L, as assessed by flow cytometry, were similar in all types of eosinophil donors, and was not altered by cytokine priming with IL-5 or GM-CSF, nor with IL-3 for basophils. By flow cytometry human basophils expressed several Siglecs (Siglec-3 > Siglec-5 > Siglec-8; Siglec-10 undetectable), similar to levels expressed on HMC-1 cells. For eosinophils, surface expression was Siglec-8 > Siglec-3, with both Siglec-5 and Siglec-10 undetectable. Conclusion Many CD33-subfamily Siglecs are expressed by human eosinophils, basophils and mast cells. Further studies are needed to explore the functional consequences of these observations.