Magda Babina

Mast cells as initiators of immunity and host defense

Abstract: Until recently, mast cells have been viewed primarily as harmful because of their key role as effector cells of allergic and potentially lethal anaphylactic reactions. Their contribution to human health appeared instead to be limited to the elimination of parasites. There is, however, growing evidence for additional beneficial functions of mast cells, particularly regarding the initiation of acquired immune reactions. Thus, mast cells can phagocytize diverse particles, take up antigens, and express a number of receptors, particularly MHC class I and II antigens, ICAM‐1 and ‐3, CD43, CD80, CD86 and CD40L which allow them to interact with T and B lymphocytes. They can also secrete numerous cytokines that induce and enhance recruitment and functions of lymphocytes. Finally, there is good evidence that mast cells present e.g. pollen and bacterial antigens, respond to bacterial superantigens, but fail to react to endogenously produced antigens or superantigens. Mast cells can also activate B cells directly to produce IgE, but this activity and the ability to produce IL‐4 or IL‐13 is restricted primarily to basophil leukocytes and mucosal mast cells. Finally, recent evidence attributes a pivotal role to the cells in natural immunity to bacteria. There is also emerging evidence that mast cells can downmodulate the immune response. While these data require further clarification, the basic ability of mast cells to initiate innate and acquired immune reactions can no longer be questioned.

HUMAN LEUKAEMIC (HMC-1) AND NORMAL SKIN MAST CELLS EXPRESS BETA 2-INTEGRINS : CHARACTERIZATION OF BETA 2-INTEGRINS AND ICAM-1 ON HMC-1 CELLS

Mast cells are bone marrow‐derived, ubiquitous connective tissue resident cells. However, their mechanisms of migration, the distribution of immature and mature cells and their interaction with other inflammatory cells are largely unclarified. Possibly, β2‐integrins play an important role in these processes. In the present investigation, the authors studied the expression and regulation of the β2‐integrins LFA‐1 (CD11a/CD18), Mac‐1 (CD11b/CD18), p150,95 (CD11c/CD18) and of the LFA‐1/Mac‐1 counter‐receptor intercellular adhesion molecule‐1 (ICAM‐1; CD54) on leukaemic (HMC‐1 cell subclone 5C6) and on normal mature human skin mast cells. The HMC‐1 cells clearly expressed CD11a, CD18 and CD54, while expression of CD11b and CD11c was low. The apparent molecular weights were 180 kDa (CD11a), 95 kDa (CD18) and 90 kDa (CD54) as determined by Western blot analysis. Phorbol myristate acetate (PMA) induced a time‐ and dose‐dependent up‐regulation of CD11a, CD11b, CD11c, CD18 and CD54 that was inhibited by cycloheximide, suggesting a dependence on de novo protein synthesis. Enhanced expression of CD11a, CD11b, CD11c and CD18 could also be confirmed at the gene level as demonstrated by semi‐quantitative reverse transcription–polymerase chain reaction (RT–PCR). Increased expression of LFA‐1/ICAM‐1 in response to PMA was accompanied by strong enhancement of homotypic cell aggregation, suggesting that newly synthesized LFA‐1/ICAM‐1 is functionally active. In order to determine a physiologically relevant way of mast cell β2‐integrin modulation, several cytokines and chemotactic mediators (interleukin‐4, IL‐4; nerve growth factor β, NGFβ; C5a; and leukotriene B4, LTB4) were tested for their influence on adhesion molecule cell surface density. Only LTB4 was shown specifically to up‐regulate CD11a and CD18, but not CD11b or CD11c. The presence of CD11a, CD11c and CD18 could be confirmed on a low percentage of normal skin mast cells by immunofluorescence, using a double staining technique. In comparison to normal skin, a significantly higher percentage of CD18+ mast cells was found in inflammatory dermatoses such as psoriasis vulgaris, atopic dermatitis and lichen planus. Therefore, mast cell β2‐integrins possibly play an important role during homing of immature mast cells as well as during the interaction of activated mast cells with other inflammatory cells.

Retinoic acid up-regulates myeloid ICAM-3 expression and function in a cell-specific fashion—evidence for retinoid signaling pathways in the mast cell lineage

Investigation of mast cell responsiveness toward retinoic acid (RA) revealed selective promotion of ICAM‐3 expression in the human mast cell line HMC‐1. This process was dose‐ and time‐dependent and detectable by flow cytometry, Western blot analysis, ELISA, and Northern blot analysis. ICAM‐3 modulation was found to be cell‐type dependent, detectable also for HL‐60 cells and monocytes but not U‐937 and only weakly for KU812 cells. Terminally differentiated skin mast cells also failed to up‐modulate their ICAM‐3, suggesting the requirement for some degree of immaturity for the process. RA‐mediated effects on ICAM‐1 expression, studied in parallel, were clearly distinct from those on ICAM‐3. Investigation of retinoid receptor expression, known to mediate intracellular RA signaling, revealed presence of RARα, RARγ, RXRβ, and RXRγ transcripts in all cell lines studied, and HMC‐1 cells were the only line lacking RXRα. RARβ, not expressed at baseline, was induced by RA in a fashion obviously correlating with ICAM‐3 up‐regulation. Increased ICAM‐3 expression was of functional significance, such that processes stimulated or co‐stimulated via ICAM‐3 (homotypic aggregation, IL‐8 secretion) were clearly enhanced upon RA pretreatment, suggesting that RA may contribute via hitherto unrecognized pathways to immune function and host defense.

LEUKOSIALIN (CD43) IS PROTEOLYTICALLY CLEAVED FROM STIMULATED HMC-1 CELLS

Leukosialin (CD43), the major sialoprotein on circulating leukocytes, has been previously described to be down-regulated on neutrophils following activation with phorbol myristate acetate (PMA). The other single cells previously examined, blood lymphocytes, do not down-regulate CD43 when stimulated by PMA. Recently, we have characterized leukosialin on the human mast cell line HMC-1 and observed that leukosialin is down-regulated after stimulation with PMA. In the present study, we have investigated the mechanism of PMA-mediated down-regulation of CD43 on HMC-1 cells (subclone 5C6). PMA caused the release of soluble leukosialin (123 kD) during HMC-1 cell activation. The molecular weight of soluble leukosialin was nearly identical to that of the cell-membrane bound molecule, suggesting a cleavage proximal from the cell membrane. Inhibitors of serine proteases, like phenylmethylsulphonyl fluoride (PMSF), benzamidine and 3, 4-dichloroisocoumarin, blocked the PMA-mediated cleavage of CD43. In all experiments, the inhibition of CD43-down-regulation was dependent on the concentration of protease inhibitors. Treatment of HMC-1 cells with various proteases (trypsin, alpha-chymotrypsin, elastase, papain, nagarse) substantially decreased anti-CD43 binding capacity and caused the release of soluble leukosialin (116 kD) or its fragments into the supernatant. Pretreatment of HMC-1 cells with neuraminidases from Vibrio cholerae or Arthrobacter ureafaciens resulted in an increased sensitivity of CD43 against proteases, whereas the effects of PMA were not influenced. In conclusion, proteolytic cleavage of CD43 is described for the first time in a cell other than neutrophils, namely HMC-1 cells. Our results suggest that serine proteases are involved in the PMA-mediated down-regulation of leukosialin on HMC-1 cells.

All-trans retinoic acid down-regulates expression and function of β2 integrins by human monocytes: opposite effects on monocytic cell lines

All‐trans retinoic acid (ATRA) plays an important role in the differentiation of malignant myeloid cells but its effects on primary leukocytes have been poorly investigated. We report here that ATRA negatively affects expression and function of leukocyte integrins that play a key role in monocyte adhesive interactions. As evidenced by flow cytometry, ATRA (at 1u2004μM) clearly and donor‐independently suppressed the expression of all integrin chains investigated (CD11a, CD11b, CD11c, and CD18), most strikingly of CD11a. Down‐regulation was detectable after 24 and maximal after 72–96 h. Reverse transcription‐PCR analysis revealed diminished steady‐state concentrations of α specific transcripts but not of the common β chain, suggesting that heterodimer expression was predominantly regulated through α chains. Results obtained with blood‐derived monocytes were in sharp contrast to those for the leukemic cell lines THP‐1 and U937, both of which showed marked increase in all integrin subunits in response to ATRA. ATRA‐pretreated monocytes displayed significantly diminished β2 integrin‐dependent homotypic aggregation, and adhesion to stimulated endothelial cells (EC), while ATRA‐pretreated monocytic cell lines showed the opposite behavior displaying markedly enhanced aggregation and CD18‐mediated adhesion to EC. Therefore, the level of leukocyte integrins was obviously a decisive factor for these adhesive interactions irrespective of the cellular source. Collectively, our data indicate a striking difference between leukemic cell lines and normal hematopoietic cells with regard to ATRA responsiveness. By acting on key adhesive structures of normal leukocytes, ATRA mediates processes that may be of substantially broad range applying to inflammation and immunity in addition to differentiation and proliferation.

ICAM-3 (CD50) is expressed by human mast cells: Induction of homotypic mast cell aggregation via ICAM-3

Intercellular adhesion molecule-3 (ICAM-3, CD50), an adhesion receptor of the immunoglobulin superfamily, is suggested to play a key role in adhesive cellular interactions during the initial phase of an immune response. We here provide evidence that ICAM-3 is abundantly expressed by cells of the human mast cell line HMC-1 and, to a lower degree, by purified skin mast cells, as demonstrated by flow-cytometry, ELISA and RT-PCR. ICAM-3 immunoprecipitated from surface biotinylated HMC-1 cells migrates as a broad band of Mr 124,000 by Western blot analysis. We also demonstrate that monoclonal antibodies directed against ICAM-3 are capable of inducing rapid HMC-1 cell aggregation, the extent of which strongly depends on the epitope recognized by the mAb applied. Interestingly, although inhibitable by two of six mAbs against LFA-1, HMC-1 aggregation induced via ICAM-3 appears to be mediated by an adhesive pathway independent of LFA-1. Dermal mast cells are also aggregated with anti-ICAM-3 mAbs, a phenomenon which has not been described before for isolated tissue mast cells. However, this process displays slower kinetics, as compared to HMC-1 cells. That anti-ICAM-3 mAbs are able to mediate biological effects is further illustrated by their capability to increase stimulation-dependent release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 from HMC-1 cells. Taken together, these results indicate that ICAM-3 is not only expressed by immature and mature human mast cells, but also possesses functional relevance and may therefore play a significant role in mast cell associated processes.

Allergic FcεRI- and pseudo-allergic MRGPRX2-triggered mast cell activation routes are independent and inversely regulated by SCF

While allergic mast cell (MC) degranulation occurs by FcεRI aggregation and varies in strength among subjects, the analogous pseudo‐allergic route was recently uncovered to proceed via MRGPRX2. Here, we examine interindividual variability in skin MC responses to FcεRI triggering vs those evoked by MRGPRX2. While population‐based variability is comparable between the routes, FcεRI‐ and MRGPRX2‐stimulated pathways are completely independent from each other, and responsiveness to one has therefore no predictive value for the other. Conversely, degranulation triggered by compound 48/80 is highly correlated to the process elicited by substance P. MRGPRX2 mRNA shows pronounced population‐based variability (coefficient of variation 102.9%). Surprisingly, stem cell factor (SCF) as the MC‐supportive mediator par excellence potently inhibits pseudo‐allergic degranulation, while it simultaneously promotes allergic stimulation via FcεRI. We conclude that SCF can have selective MC‐dampening functions. Clinically, the data imply that subjects highly reactive in one pathway are not automatically hyper‐responsive in terms of the alternative route.