1. Mast Cell Receptors Prof.Dr. Mona Gamal Dr. Mohammed Nabil Created by
Under supervision of
Prof.Dr. Mona Gamal

2. Fc receptors
An Fc receptor is a protein found on the surface of certain cells - including natural killer cells, macrophages, neutrophils, and mast cells - that contribute to the protective functions of the immune system.
Its name is derived from its binding specificity for Fc region of antibodies that are attached to infected cells or invading pathogens.
Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity

4. Classes of Fc receptors:
There are several different types of Fc receptors, which are classified based on the type of antibody that they recognize. For example:
Those that bind IgG, are called Fc-gamma receptors (FcγR).
Those that bind IgA are called Fc-alpha receptors (FcαR).
Those that bind IgE are called Fc-epsilon receptors (FcεR).

5. 1) Fc-gamma receptors:
All Fcγ receptors (FcγR) are the most important Fc receptors for inducing  phagocytosis  of  opsonized (coated) microbes. This family includes several members, FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), which differ in their antibody affinities due to their different molecular structure. FcγRI binds to IgG more strongly than FcγRII or FcγRIII. FcγRI also has an extracellular portion composed of three immunoglobulin (Ig)-like domains, one more domain than FcγRII or FcγRIII has. This property allows activation of FcγRI by a sole IgG molecule (or monomer), while the latter two Fcγ receptors must bind multiple IgG molecules within an immune complex to be activated.

6. Another FcR is expressed on multiple cell types and is similar in structure to MHC class I. This receptor also binds IgG and is involved in preservation of this antibody. However, since this Fc receptor is also involved in transferring IgG from a mother either via the placenta to her fetus or in milk to her suckling infant, it is called the neonatal Fc receptor (FcRn). Recently, research suggested that this receptor plays a role in the homeostasis of IgG serum levels.

7. 2) Fc-alpha receptors:
Only one Fc receptor belongs to the FcαR subgroup, which is called FcαRI (or CD89). FcαRI is found on the surface of neutrophils, eosinophils, monocytes, some macrophages (including Kupffer cells), and some dendritic cells. It is composed of two extracellular Ig-like domains. It signals by associating with two FcRγ signaling chains. Another receptor can also bind IgA, although it has higher affinity for another antibody called IgM. This receptor is called the Fc-alpha/mu receptor (Fcα/μR) and is a type I transmembrane protein. With one Ig-like domain in its extracellular portion, this Fc receptor is also a member of the immunoglobulin superfamily.

8. 3) Fc-epsilon receptors:
Two types of FcεR are known:
a) High-affinity receptor FcεRI is a member of the immunoglobulin superfamily (it has two Ig-like domains). FcεRI is found on epidermal Langerhans cells, eosinophils, mast cells and basophils. As a result of its cellular distribution, this receptor plays a major role in controlling allergic responses. FcεRI is also expressed on antigen-presenting cells, and controls the production of important immune mediators called cytokines that promote inflammation.
b) Low-affinity receptor FcεRII (CD23) is a C-type lectin. FcεRII has multiple functions as a membrane-bound or soluble receptor; it controls B cell growth and differentiation and blocks IgE-binding of eosinophils, monocytes, and basophils.

9. Functions of Fc receptors
Fc receptors are found on a number of cells in the immune system including phagocytes like macrophages and monocytes, granulocytes like neutrophils and eosinophils, and lymphocytes of the innate immune system (natural killer cells) or adaptive immune system (e.g., B cells). They allow these cells to bind to antibodies that are attached to the surface of microbes or microbe infected cells, helping these cells to identify and eliminate microbial pathogens. The Fc receptors bind the antibodies at their Fc region (or tail), an interaction that activates the cell that possesses the Fc receptor. Activation of phagocytes is the most common function attributed to Fc receptors. For example, macrophages begin to ingest and kill an IgG-coated pathogen by phagocytosis following engagement of their Fcγ receptors.

10. Another process involving Fc receptors is called antibody-dependent cell-mediated cytotoxicity (ADCC). During ADCC, FcγRIII receptors on the surface of natural killer (NK) cells stimulate the NK cells to release cytotoxic molecules from their granules to kill antibody-covered target cells.
FcεRI has a different function. FcεRI is the Fc receptor on granulocytes, that is involved in allergic reactions and defense against parasitic infections. When an appropriate allergic antigen or parasite is present, the cross-linking of a least two of IgE molecules and their Fc receptors on the surface of a granulocyte will trigger the cell to rapidly release preformed mediators from its granules.

12. Cellular activation by Fc Receptors
Fc receptors recognize microbes that have been bound by antibodies. The interaction between the bound antibodies and the cell surface Fc receptor activates the immune cell to kill the microbe. This example shows the phagocytosis of an opsonized microbe.

13. Fc receptors on mast cells
IgE antibodies bind to antigens of allergens. These allergen-bound IgE molecules interact with Fcε receptors on the surface of mast cells. Activation of mast cells following engagement of FcεRI results in a process called degranulation, whereby the mast cell releases preformed molecules from its cytoplasmic granules; these are a mixture of compounds including histamine, proteoglycans, and serine proteases. Activated mast cells also synthesize and secrete lipid-derived mediators (such as prostaglandin, leukotrienes, and platelet-activating factor) and cytokines (such as interleukin 1, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 13, tumor necrosis factor-alpha, GM-CSF, and several chemokines. These mediators contribute to inflammation by attracting other leukocytes.

14. Activation of mast cell degranulation by IgE interaction with FcεRI
Activation of mast cell degranulation by IgE interaction with FcεRI. 1 = antigen; 2 = IgE; 3 = FcεRI; 4 = preformed mediators (histamine, proteases, chemokines, heparin); 5 = granules; 6 - mast cell; 7 - newly-formed mediators (prostaglandins, leukotrienes, thromboxanes, platelet-activating factor)

15. Toll-like Receptors Toll-like Receptors Regulate Mast Cell Responses
Increasing evidence suggests that mast cells play an important role in TLR-mediated responses, e.g. host defence against Gram-negative bacterial infection. Optimal protective effects conferred by mast cells require a functional TLR4, and are mediated through the release of pro-inflammatory mediators, particularly TNF-α. Besides TLR4, murine mast cells also express TLR2 and TLR6, suggesting that they play a protective role against other pathogens.

16. Whereas recent studies examining allergic diseases focus on TLRs on dendritic cells and T regulatory cells, the importance of TLRs on mast cells needs to be further investigated. Studies analysing TLR signaling on mast cell function have yielded heterogeneous results. Lipopolysaccharide stimulation of mast cells through TLR4 induces the production of inflammatory cytokines, e.g. IL-1β, TNF-α, IL-6 and IL-13, without mast cell degranulation, suggesting that mast cells can release cytokines independent of their classic degranulation pathway. Although the study reports increased IL-13 secretion with lipopolysaccharide stimulation, there is no increase in IL-4 and IL-5, two other cytokines important in promoting allergic inflammation. In a different study, lipopolysaccharide stimulation combined with IgE cross-linking, increased the production of the Th2-associated cytokines IL-5, IL-10 and IL-13. As concurrent lipopolysaccharide and allergen exposure may cause heightened allergic inflammation in certain situations, the effects of TLR4 signaling on mast cell cytokine production may be an area of therapeutic interest.

17. In addition to TLR4, other TLRs may be critical in the interface of innate and adaptive immunity. Stimulation with the TLR2 ligand peptidoglycan produced different murine mast cell responses when compared with lipopolysaccharide exposure. Peptidoglycan causes mast cell degranulation as well as the production of IL-4 and IL-5. Therefore, specific TLRs on mast cells appear to signal via different pathways.
The expression of TLRs on human mast cells was examined using human cord blood-derived mast cells (CBMCs). TLR1, TLR2 and TLR6, but not TLR4 expression is demonstrated on CBMCs. The stimulation of CBMCs with lipopolysaccharide does not produce significant increases in granulocyte macrophage-colony-stimulating factor or IL-1β. However, priming mast cells with IL-4 and soluble CD14 induces the production of TNF-α, IL-5, IL-10 and IL-13 after lipopolysaccharide exposure. An allergic Th2 response with IL-4 production may increase the expression of TLR4 on mast cells, a finding previously shown in human B cells.

18. Allergen induction via dendritic cells and T cells may prime mast cells to express TLR4, and subsequent endotoxin exposure may lead to increased allergic inflammation. These interactions contrast with the potential protective effect of endotoxin exposure on dendritic cells or T regulatory cells. Investigation of these potentially conflicting pathways may offer insights into mast cell function during innate and adaptive immunity.

19. Sialic acid-binding immunoglobulin-like lectin(Siglec) family
The immunoglobulin superfamily is composed of a large number of cell surface proteins that play a vital role not only in immunity, but also in controlling the behavior of cells in different tissues through their abilities to
mediate cell surface recognition events.
Siglecs are a recently defined subset of this superfamily. This group of adhesion receptors contains variable numbers of extracellular C2-set Ig domains (two to seventeen) and a unique N-terminal variable region (V-set) Ig domain that confers the ability to bind to sialic acid containing carbohydrate groups (sialosides) of glycoproteins and glycolipids. With the exception of Siglec1 and 4, the cytoplasmic domains of the other 9 Siglecs contain one or more ITIM or ITIM-like motifs that are often dynamically phosphorylated and are characteristic of accessory proteins that negatively regulate transmembrane signaling of cell surface receptor proteins.

20. These properties suggest that Siglecs may be involved in cell-cell interactions and regulation of cellular activation within the immune system resulting in intracellular inhibitory signals.
The initial discovery of this lectin family came about through independent studies on a sialoadhesin (Siglec1), a macrophage lectin-like adhesion, as well as on the studies of Siglec2 and 3. Both Siglec2 and 3 are the members of Ig superfamily restricted on B cell and both play an important role in regulating B cell activation through cell-cell interactions. Over the past several years a cluster of genes,
that encode novel Siglecs that are highly related to Siglec3 (CD33), has been identified and mapped to the long arm (q13.4) of chromosome 19

21. This cluster includes Siglec3, 5, 6, 7, 8, 9 and 10 which share a high degree (50-80%) of sequence similarities. Siglecs are mainly expressed in cells of the hematopoietic origin; ten of the eleven known Siglec family members are expressed among the cell lineages of the hematopoietic system while only two are expressed in neuronal cells.
Both B cells and monocytes express a relatively large number of Siglecs, for instance, Siglec2, 5, 6, 9, 10 are expressed on the surface of B cells, while Siglec3, 5, 7, 9, 10 are expressed in monocytes. Siglec9 is broadly expressed in at least five different cell types: neutrophils, monocytes, NK cells, B cells, and subsets of CD8 positive T cells.

22. It is of interest to note that a relatively high level of expression of Siglec5, 6 and 8 proteins can be detectedon MC surface during allergic inflammation. The long form of Siglec8 consists of three Ig domains in the extracellular domain and contains one ITIM motif in the cytoplasmic tail. Interestingly, Siglec8 appears to be preferentially expressed in eosinophils, MCs and basophils.
Although it has not been reported whether Siglec5, 6 or 8 inhibit FcεRI mediated MC degranulation, the fact that cross-linking of Siglec8 by an antibody can dampen cell activation and reduce eosinophil viability through induction of apoptosis suggest that these receptors may provide an endogenous negative signal in MCs in vivo.

23. Mast cell function-associated antigen (MAFA)
MAFA is also known as killer cell lectin-like receptor subfamily G member 1 (KLRG1) which is a type II transmembrane protein containing a C-type lectin carbohydrate recognition domain, an intracellular ITIM- like motif, and 4 potential N-glycosylation sites. Human MAFA gene contains 5 exons with 2 alternatively
spliced variants. One lacks exon 3 (E3-minus) and the other lacks both exons 3 and 4 (E3/4-minus). E3-minus MAFA is predicted to have a short extracellular region and its expression appears to be restricted to lung MCs and basophils.

24. Activities of MAFA have been studied mostly using rat RBL-2H3 cell line. Studies using immunofluorescence, fluorescent resonance energy transfer and time-resolved phosphorescence anisotropy measuring the rotational mobility of both MAFA and FcεRI provided evidence for the direct interaction of MAFA and FcεRI. MAFA clustering by its specific monoclonal antibody has been shown to inhibit both the FcεRI- mediated hydrolysis of phosphatidyl phosphoinositides and the transient rise in the intracellular concentration of free calcium ion resulting in suppressed secretory responses and cytokine synthesis. MAFA clustering however did not affect degranulation induced by the Ca2+ ionophore A23187 which bypass the receptor and the upstream
signaling event, suggesting that inhibition mediated by MAFA may be due to coupling to a cascade upstream of PLC-γ activation. This inhibitory action is initiated by tyrosine phosphorylation of MAFA’s ITIM motif through SHP2 or SHIP but not SHP1. Interestingly the MAFA mediated inhibition of the activation of the activating receptor (FcεRI) does not require colligation of the activating and inhibitory receptors.