Mast cells and complement system: Ancient interactions between components of innate immunity.

The emergence and evolution of the complement system and mast cells (MCs) can be traced back to sea urchins and the ascidian Styela plicata, respectively. Acting as a cascade of enzymatic reactions, complement is activated through the classical (CP), the alternative (AP), and the lectin pathway (LP) based on the recognized molecules. The system's main biological functions include lysis, opsonization, and recruitment of phagocytes. MCs, beyond their classic role as master cells of allergic reactions, play a role in other settings, as well. Thus, MCs are considered as extrahepatic producers of complement proteins. They express various complement receptors, including those for C3a and C5a. C3a and C5a not only activate the C3aR and C5aR expressing MCs but also act as chemoattractants for MCs derived from different anatomic sites, such as from the bone marrow, human umbilical cord blood, or skin in vitro. Cross talk between MCs and complement is facilitated by the production of complement proteins by MCs and their activation by the MC tryptase. The coordinated activity between MCs and the complement system plays a key role, for example, in a number of allergic, cutaneous, and vascular diseases. At a molecular level, MCs and complement system interactions are based on the production of several complement zymogens by MCs and their activation by MC-released proteases. Additionally, at a cellular level, MCs act as potent effector cells of complement activation by expressing receptors for C3a and C5a through which their chemoattraction and activation are mediated by anaphylatoxins in a paracrine and autocrine fashion.

Crosstalk Between Mast Cells and Adipocytes in Physiologic and Pathologic Conditions.

Excessive fatty acids and glucose uptake support the infiltration of adipose tissue (AT) by a variety of immune cells including neutrophils, pro-inflammatory M1 macrophages, and mast cells (MCs). These cells promote inflammation by releasing pro-inflammatory mediators. The involvement of MCs in AT biology is supported by their accumulation in the AT of obese individuals along with significantly higher serum levels of MC-derived tryptase. AT-resident MCs under the influence of locally derived adipokines such as leptin become activated and release pro-inflammatory cytokines including TNFα that worsens the inflammatory state. MCs support angiogenesis in AT by releasing chymase and inducing preadipocyte differentiation and also the proliferation of adipocytes through 15-deoxy-delta PGJ2/PPARγ interaction. Additionally, they contribute to the remodeling of the AT extracellular matrix (ECM) and play a role in the recruitment and activation of leukocytes. MC degranulation has been linked to brown adipocyte activation, and evidence indicates an important link between MCs and the appearance of BRITE/beige adipocytes in white AT. Cell crosstalk between MCs and AT-resident cells, mainly adipocytes and immune cells, shows that these cells play a critical role in the regulation of AT homeostasis and inflammation.

Significance of mast cells in spermatogenesis, implantation, pregnancy, and abortion: Cross talk and molecular mechanisms.

Both subsets of MCs including MCTC (tryptase-positive, chymase-positive) and MCT (tryptase-positive, chymase-negative) are present in the testis and epididymis. Increased number of MCs, higher levels of MC-released tryptase in testis and seminal plasma of males with fertility problems, and promoting sperm motility in individuals with oligozoospermia after using MC blockers provide evidence that MCs may play a role in male infertility/subfertility disturbances. MC-released tryptase and histamine contribute to the fibrosis and may disrupt spermatogenesis. MCs not only influence the process of spermatogenesis but also have effects on the function of other testis-residing cells. MC-derived histamine may influence the steroidogenesis of Leydig cells by acting through H1R and H2R receptors. Additionally, the interaction between MC-released ATP and P2X receptors expressed on the peritubular cells may induce the production of the pro-inflammatory mediators by peritubular cells. Further investigations showed that MCs may be involved in the pathology of female infertility during implantation, pregnancy, and abortion. In the uterus, MCT subtype is abundant in myometrium and adjacent basal layer while MCTC subtype is distributed in all layers. MCs in response to hormones mainly estradiol and progesterone become activated and release a wide range of mediators including histamine, VEGF, proteases, and metalloproteinases (MMPs) that have a role in different stages of pregnancy. An increasing influx of MCs to the cervix during the pregnancy occurs that helps to the physiologic cervical ripening. While MMPs degrade the extracellular matrix (ECM), VEGF modulates neovascularization and histamine influences the embryo implantation. MC-derived histamine may have a positive effect during implantation due to its participation in tissue remodeling. MC proteases including tryptase and chymase activate the precursors of MMP2 and MMP9 to mediate ECM degradation during the physiologic menstrual cycle. There is a line of evidence that MCs have a role in abortion by releasing TNF-α.

Immunology of Bee Venom.

Bee venom is a blend of biochemicals ranging from small peptides and enzymes to biogenic amines. It is capable of triggering severe immunologic reactions owing to its allergenic fraction. Venom components are presented to the T cells by antigen-presenting cells within the skin. These Th2 type T cells then release IL-4 and IL-13 which subsequently direct B cells to class switch to production of IgE. Generating venom-specific IgE and crosslinking FcεR1(s) on the surface of mast cells complete the sensitizing stage in allergic individuals who are most likely to experience severe and even fatal allergic reactions after being stung. Specific IgE for bee venom is a double-edged sword as it is a powerful mediator in triggering allergic events but is also applied successfully in diagnosis of the venom allergic patient. The healing capacity of bee venom has been rediscovered under laboratory-controlled conditions using animal models and cell cultures. The potential role of enzymatic fraction of bee venom including phospholipase A2 in the initiation and development of immune responses also has been studied in numerous research settings. Undoubtedly, having insights into immunologic interactions between bee venom components and innate/specific immune cells both locally and systematically will contribute to the development of immunologic strategies in specific and epitope-based immunotherapy especially in individuals with Hymenoptera venom allergy.

Cloning and Stable Expression of cDNA Coding For Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1, CD31) in NIH-3T3 Cell Line.

PURPOSE: PECAM-1 (CD31) is a glycoprotein expressed on endothelial and bone marrow precursor cells. It plays important roles in angiogenesis, maintenance and integration of the cytoskeleton and direction of leukocytes to the site of inflammation. We aimed to clone the cDNA coding for human CD31 from KG1a for further subcloning and expression in NIH-3T3 mouse cell line. METHODS: CD31 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 µg of recombinant construct and 6 µl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. RESULTS: 2235 bp specific band was aligned completely to human CD31 reference sequence in NCBI database. Transient and stable expression of human CD31 on transfected NIH-3T3 mouse fibroblast cells was achieved (23% and 96%, respectively) as shown by flow cytometry. CONCLUSION: Due to murine origin of NIH-3T3 cell line, CD31-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD31, with no need for purification of recombinant proteins.

Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line.

BACKGROUND: CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. METHODS: Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. RESULTS: Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively). CONCLUSION: Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line.

PURPOSE: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. METHODS: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 µg of recombinant construct and 6 µl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. RESULTS: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. CONCLUSION: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems.