Connective Tissue PDF - 2020/2021

2020/2021 Isabella Ronchi Connective tissue Connective tissue is composed by different cell types deriving from mesenchyme cells of mesodermal origin. There are three types of connective...

2020/2021 Isabella Ronchi Connective tissue Connective tissue is composed by different cell types deriving from mesenchyme cells of mesodermal origin. There are three types of connective tissue: o Connective tissue proper: mucous, loose, dense, adipose o Supportive connective tissue: cartilage, bone o Liquid connective tissue: blood and lymphoid organs Connective tissue contains cells, fibres and an abundant extra-cellular matrix (ECM). It’s made of fibrillary components (collagen fibres, reticular fibres and elastic fibres) and ground substance (water, electrolytes, glycoproteins, glycosaminoglycans, proteoglycans and enzymes). The cells The resident cells of the connective tissue include: o Fibroblasts: they are the principal cells of the connective tissue, responsible for the synthesis of collagen, elastic and reticular fibres and the carbohydrates of the ECM. o Macrophages (or histiocytes): they are derived from the bone marrow progenitors during embryogenesis, and from circulating monocytes extra-vasated in tissues. They have a defence function. They phagocytose microbes, damaged and aged cells, cell debris, ECM components; they present antigens during immune response; they secrete cytokines and enzymes. When they are inactive, they also have homeostatic functions, including: trophic functions in tissues, important for healing after tissue damage and during embryogenesis for the development of all other tissues. After activation, as a consequence of an inflammatory process, macrophages detach from collagen fibres and become migrating or activated macrophages, moving by ameboid movement and performing an intense phagocytic activity. They are about 10-30 µm in diameter and they can have different shapes: spindle or stellate. They have a kidney shaped nucleus and abundant lysosomes, RER, Golgi apparatus and cytoskeleton. Their life span is about 2-3 months and they have a low rate of proliferation. When they’re in their resting state, they’re associated with collagen fibres by adhesion. Liver Kupffer cells Spleen Red pulp macrophages Peritoneal cavity Peritoneal macrophages Lung Alveolar macrophages (dust cells) Bone Osteoclasts Central nervous system Microglia o Adipocytes: the nucleus and the thin cytoplasm are displaced peripherally, below the plasma membrane as a consequence of the continuous fat accumulation. 2020/2021 Isabella Ronchi o Mast cells: they arise from the bone marrow precursor stem cells circulating in the bloodstream and then enter the connective tissue where they differentiate in mast cells and perform their task. Their cytoplasm is rich in granules containing mediators of inflammation (e.g., histamine, heparin, serine protease, leukotrienes). They are around 20-30 µm in diameter. They have IgE receptors and function in mediating the inflammatory process and immediate hypersensitivity reactions and allergy. The transient cells of the connective tissue are: o Leukocytes (neutrophils, lymphocytes, monocytes, eosinophils, basophils): they exit the bloodstream and move in the connective tissue by crossing the walls of small veins and capillaries. Their number increases during inflammation. They move by diapedesis, a finger-like protrusion or a pseudopodium penetrates between two endothelial cells. o Plasma cells: they’re large ovoid cells, around 20 µm in diameter. They derive from B lymphocytes and they’re responsible for antibody synthesis and secretion. Their life span is around 2-4 weeks. Plasma cell nuclei contain heterochromatin radiating out from the centre, thus appearing as a “cartwheel” or as a “clock face”. They have a well-developed RER and they produce immunoglobins. The fibres All types of fibres are produced by fibroblasts. There are several types of fibres: o The collagen fibres are the most abundant. They have a certain degree of flexibility, but their specific feature is the high tensile strength, therefore they have great ability to resist to mechanical stress. This is important for tissue support, connection and flexibility. Collagen fibres are long and non-branched. Sometimes they have collagen crimps (foldings, curves), especially common in the tendons. There’s a close relationship between crimp architecture and biomechanical properties. There are about 25 types of collagen, in particular o I – skin, bone, tendon, cornea (most common type) o II – cartilage o III – reticular fibres, loose connective tissue, blood vessel walls, dermis o IV – basal lamina of epithelium and kidney glomeruli Collagen is mainly produced by fibroblasts, but also by osteoblasts, chondrocytes and pericytes. Collagen is synthesized in the RER and assembled in the EER. The folded pro-collagen molecules pass to the Golgi apparatus, where the fibrils assemble and are kept together by covalent cross-linking. Protocollagen is secreted outside the cells and converted to collagen by peptidases. It’s assembled into fibrils and fibres in the extra-cellular space. Collagen fibres are formed by fibrils. Diameter of the fibres is 1-2 µm, depending on the number of fibrils. Collagen is the most abundant protein in the human body (30% of dry weight). Several pathological conditions related to mutations or altered collagen turnover exist (e.g., osteogenesis imperfecta, progressive systemic sclerosis and fibrosis). 2020/2021 Isabella Ronchi o The reticular fibres are small, branched fibres filling spaces and providing the thin stroma of organs. They’re made of type III collagen. Reticular fibres make a loose 3D network and they’re around 0,2-1 µm in diameter. They’re common in the liver, spleen and lymph nodes. The prevalence of reticular fibres is also an indicator of tissue immaturity. For example, they’re prominent in the initial stages of wound healing, where they provide early mechanical strength to the newly synthesized ECM. They are gradually replaced by stronger type I collagen fibres. o The matrix fibres are intermediate fibres made of elastin. They’re branched fibres that allow tissues to respond to stretch and distension. Elastic fibres are produced by fibroblasts and vascular smooth muscle cells. They are formed by two components: amorphous + microfibrillary (intermixed with collagen fibres). The amorphous component is elastin. Desmosine and isodesmosine are amino acids unique to elastin, allowing covalent bonding of elastin molecules. The microfibrillary component surrounds the central core and contains microfibrils formed by fibrillin subunits. Marfaan syndrome is a genetic disease caused by dominant mutations in the Fibrillin-1 gene. It produces deficiency of elastin-associated microfibrils, resulting in skeletal, ocular and cardiovascular defects. Elastin may be degraded by the enzyme elastase, a proteolytic enzyme. α1-antitrypsin inhibits proteolytic enzymes, including elastase, and its activity is important to prevent local tissue injury. Deficiency of α1-antitrypsin leaves elastin fibres in alveolar walls unprotected. The destruction of the connective tissue of alveolar walls causes an inefficiency in elastic expansion of the alveoli (emphysema) and may cause an acute collapse. The Extra Cellular Matrix The main functions of the Extra Cellular Matrix are: o Binding and packing of tissues o Connecting, anchoring and supporting the body and its organs o Transporting metabolites between capillaries and tissues o Repairing injuries via cell proliferation and fibre formation 2020/2021 Isabella Ronchi Ground substance is rich in proteoglycans (core proteins + glycosaminoglycans) and multiadhesive glycoproteins (fibronectin and laminin). Glycoproteins are proteins conjugated to saccharides lacking a serial repeat unit (protein >> carbohydrate). Proteoglycans are proteins conjugated to polysaccharides with a serial repeat unit (carbohydrate >> protein). Glycosaminoglycans are long polysaccharides containing a repeating disaccharide unit. The disaccharide units contain N- acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc) and an acidic sugar (glucuronic acid). GAGs are highly negatively charged, highly hydrated molecules. They are viscous and have low compressibility. They act as lubricants and mechanical shock absorbers. GAGs of physiological significance include: o Hyaluronic acid (it’s unique in that it doesn’t contain any sulphate and it’s not found covalently attached to proteins as a proteoglycan) o Dermatan Sulphate o Chondroitin Sulphate o Heparin o Heparan Sulphate o Keratan Sulphate Fibronectin links cells to several components of the ECM. It’s recognised by cells as a ligand for cell surface adhesive receptors (integrin family). It’s responsible for cell adhesion and involved in cell migration. Laminin binds to components of the ECM (collagen). Cells recognise the basement membrane through receptors that interact primarily with laminin. Several proteases are involved in the degradation of ECM components. Matrix Metalloproteinases are involved in ECM remodelling, in physiological (morhogenesis, wound healing) and pathological conditions (inflammation, atherosclerosis, tumour invasion and fibrogenesis). Their function is activated by the presence of zinc. 2020/2021 Isabella Ronchi The main functions of connective tissue proper are: o Connection of different tissues to maintain o Protection of delicate organs a functional integration o Trophic function o Structural support o Defence function o Serving as a medium for exchange and o Repair of damaged tissue regulating metabolites diffusion There are several types of connective tissue proper: o Mucous connective tissue (Warthon’s jelly) is loose and amorphous. It’s found in the umbilical cord, in aqueous humour and in the dental pulp. o Loose connective tissue (areolar) has less fibres and more ECM. It’s not as resistant to stress and it has a viscous, gel- like consistency. The loose connective tissue contains capillary beds throughout the body and it’s important for the diffusion and exchange of waste and nutrients. Most of the cells present in the loose connective tissue are transient and migrate from local blood vessels. The loose connective tissue is the site of inflammatory and immune reactions. It’s found in: lamina propria of the mucosa and submucosa of hollow organs, in the tunica intima of blood vessels and in the stroma of solid organs. o Dense regular connective tissue: has either parallel (tendons and ligaments) or orthogonal (cornea) bundles. Dense connective tissue has more fibres and less ECM and is very resistant to stress. Collagen fibres are in parallel bundles with fibroblasts and the connective tissue cells are squeezed between the thick fibres. o Dense irregular connective tissue has randomly-arranged fibres (interwoven). It can be capsular (external capsule of solid organs) or lamellar (Pacinian corpuscles, skin sensor receptors). This tissue can resist tensile forces from any direction. Finally, adipose tissue is formed by adipocytes or fat cells, enveloped by a basal lamina and sustained by loose connective tissue. There are two types of adipose tissue: unilocular (or white) and multilocular (or brown). 2020/2021 Isabella Ronchi UNILOCULAR (WHITE) MULTILOCULAR (BROWN) It forms the white fat in adult humans. Its abundance It’s infrequent in adults and abundant in children. depends on the nutritional status. It’s used for It’s also abundant in rodents and in hibernating storage of energetic substances, support, thermal animals. It’s used in lipid degradation and for heat insulation, hormone production and organ production (thermogenesis). In the cytoplasm protection. Adipocytes produce some important there are several droplets of fat, not just a single big hormones, among which leptin, a factor regulating droplet like in unilocular adipocytes. the energy homeostasis (inhibits food intake, stimulates metabolic rate). Cartilage is composed of cells (chondroblasts/chondrocytes) and an ECM (fibrillary components and ground substance). There are no blood vessels and no nerves, except than in the perichondrium. Cartilage has mainly skeletal functions, including: o In the embryo and in the foetus, cartilage forms the skeletal model that will be almost completely replaced by bone tissue o During development it allows the growth of the long bones (metaphyseal cartilage) o The articular cartilage is a smooth surface that allows sliding of articular surfaces o It forms the framework of the outer ear, the nose and some hollow organs of the respiratory system (trachea, larynx, bronchi) In the ECM fibres are mainly type II collagen (50% of dry weight) and type I collagen, elastic fibres. The ground substance is formed by proteoglycans (30-40% of dry weight), glycoproteins, lipids and water (80% of total weight). Cartilage is a specialised connective tissue in which the firm consistency of the ECM allows the bearing of mechanical stress without permanent distortion. The ground substance undergoes dilation under compression and collagen resists to tensile forces. When the cartilage is compressed, water diffuses out and when the compression stops the cartilage reabsorbs the water. Chondroblasts are the precursor cells, which are located in the chondrogenic layer and actively produce and secrete ECM components. Chondrocytes are the mature cells and they are located in the matrix, grouped in 2-4 cells (isogenous groups). 2020/2021 Isabella Ronchi CHONDROBLASTS CHONDROCYTES o Bigger o Smaller o Active metabolism o Intensely stained nucleus o Vesicular nucleus and evident nucleolus o Scarce cytoplasm o Vacuolised cytoplasm (lipids and glycogen) o Low metabolic activity o Lacunae (spaces between ECM and cells) o Lacunae The perichondrium is important for the nutrition of the cartilage. It consists in dense connective tissue surrounding the cartilage (except in articular cartilage). It contains blood, nerve supply, lymphatics, collagen fibres and fibroblasts. The deep layer of perichondrium, where chondroblasts are present, is called chondrogenic layer. There are two mechanisms of chondrogenesis: o Appositional growth: deposition of new cartilage at the surface of an existing cartilage, deriving from the inner layer of perichondrium. Cells in the inner layer of perichondrium differentiate in chondroblasts, producing ECM components. New fibroblasts are produced to maintain the cell population of the perichondrium. After birth, the perichondrium doesn’t produce chondroblasts, but this potential remains and may be effective upon growth stimulation. o Interstitial growth: formation of new cartilage within existing cartilage. Chondrocytes within their lacunae divide by mitosis and secrete new matrix. The two daughter cells share the same lacuna. Chondrocytes progressively move apart as they deposit in the ECM. 2020/2021 Isabella Ronchi HYALINE CARTILAGE - Most abundant cartilage - It forms the foetal skeleton; in the adult it’s restricted to few places - It supports organs of the respiratory system - Glassy appearance - It’s surrounded by the perichondrium (except articular cartilage) - Flexible and resilient It’s present in: articular surfaces, rib cartilage, tracheal rings, larynx, bronchi ELASTIC CARTILAGE - Contains many elastic fibres - Able to resist repeated bending - Yellowish opaque colour, has elastic fibres running in all directions It’s present in: external ear, auditory tubes, epiglottis FIBROCARTILAGE - Contains many collagen fibres - Resists compression and tension - An intermediate between hyaline cartilage and dense connective tissue - Type I collagen, no perichondrium - Small cells arranged in short rows - White colours, heterogeneous appearance It’s present in symphysis, intervertebral discs, meniscus of the knee 2020/2021 Isabella Ronchi Articular cartilage lacks the perichondrium, so chondroblasts progenitor cells are missing. The growth can only occur by mitosis of inner chondroblasts in the ECM (interstitial growth). The reason why there is no perichondrium is that articular cartilage needs a smooth surface to allow sliding of the articular surfaces. Collagen fibres form a 3D network linked to proteoglycans, able to bear mechanical pressure from different directions. The epiphyseal plate is a hyaline cartilage in the metaphysis at each end of a long bone. The plate is found in children and adolescents and is the site of endochondral ossification and longitudinal growth of long bones. Cartilage continually grows and is replaced by bone as quickly as it grows. The bone lengthens entirely by growth of the epiphyseal plate. During aging, cartilage ECM proteoglycans decrease and non-collagen fibres increase. This leads to an impaired hydration and to a less efficient diffusion of nutrients and hormones. Non-collagen fibres aren’t stable, and are destroyed, leading to the formation of cavities (asbestiform degeneration). Osteoarthritis is a degenerative joint disease related to injury and aging of articular cartilage. It leads to inflammation, pain, reduction of movement, joint deformity and bone destruction. The main functions of the skeletal system are: o Support against gravity o Leverage for muscle movement o Protection of soft internal organs (brain, lungs, bone marrow) o Storage for calcium and phosphorus The ECM is strongly mineralised, giving this tissue high resistance, rigidity and hardness. It’s not a static tissue, it’s continuously remodelled and renewed. Bone tissue is composed of specialised cells (2% of weight) and a strong flexible matrix made of calcium phosphate crystals and collagen fibres. The bone is also made of 10% water. If the inorganic component of the ECM is destroyed, the result is a loss of hardness and rigidity: the bone becomes flexible, but it retains its resistance to tensile forces. If, on the other hand, the organic component is destroyed, the bone shape and the original size are maintained, but it becomes fragile like porcelain. There are three types of bones: long bones, flat bones and short bones. In long bones, the extremities are called epiphysis, while the central part is called diaphysis (or shaft). There are two types of bone membrane: o The periosteum: protective membrane of connective tissue supplied with nerve fibres, blood and lymphatic vessels entering the bone. It provides anchoring points for tendons and ligaments. It’s a layer of dense connective tissue, rich in collagen fibres and fibroblasts. It’s made of two layers: outer 2020/2021 Isabella Ronchi (fibrous) and inner (osteogenic). The deep layer contains progenitor cells that can differentiate in osteoblasts. Collagen bundles that enter into the bone matrix are named Sharpey fibres (perforating fibres). o The endosteum: delicate connective membrane covering internal surfaces of bone. Covers the trabeculae of spongy bone. It lines all inner surfaces of bone cavities. It contains a single cell layer of osteoprogenitor cells. It’s thinner than the periosteum and its functions is related to bone nutrition and to provide the bone with new cells. Non-lamellar (woven) bone is an emergency tissue, made of intertwined bundles. It’s found in inferior vertebrates, foetal bone and fracture repair. Lamellar bone (mature) can be compact/cortical (outer shell of flat bones, surface of short bones and epiphysis and diaphysis of long bones) or spongy/trabecular/cancellous (short bones, epiphysis of long bones). The osteon is the structural unit of the compact bone. The lamellae are column- like matrix tubes composed of collagen and crystals of salts. Lamellae encircling the Haversian canal form the osteon, a small cylinder-shaped structure 0,9-1,2 mm high, made by a variable number of lamellae (approximately 5-20). Each lamella is characterised by regularly parallel oriented collagen fibres. Collagen properties and their particular arrangement are responsible for bone strength. Spongy bone is formed by trabeculae, which are made of irregular lamellae containing lacunae where osteocytes lay. They form a 3D structure which delimits cavities containing the bone marrow. Haversian systems are lacking: osteocytes exchange metabolites with bone marrow sinusoids by interconnected canaliculi. There are different types of cells: o Osteoprogenitor cells (undifferentiated cells) are able to divide to replace themselves and become osteoblasts. They can be found in the inner layer of the periosteum and endosteum. They are arranged in a single epithelial-like cell layer. o Bone-lining cells cover bones that aren’t remodelling. They derive from osteoblasts. Following completion of matrix formation, osteoblasts have three possible fates: become osteocytes embedded in mineralised bone matrix, die by apoptosis or transform into quiescent bone lining cells. One proposed function is removal of demineralised matrix of the bone surface prior to bone formation. 2020/2021 Isabella Ronchi o Osteoblasts form matrix and collagen fibres. They possess the receptor for the parathyroid hormone that regulates calcium metabolism. o Osteocytes are mature cells that no longer secrete matrix. During matrix mineralisation osteoblasts become osteocytes that remain trapped in lacunae and the cytoplasmic processes remain in the canaliculi. Osteocytes are important in mechanotransduction, they respond to mechanical forces by increasing or decreasing matrix stiffness. o Osteoclasts are multinucleated and polarised cells involved in bone degradation. They originate from haematopoiesis, in particular from the monocyte-macrophage lineage. Osteoclasts are activated by factors released by osteoblasts, stimulated in turn by the parathyroid hormone. Osteoclasts are inhibited by calcitonin, for which they express the receptor themselves. Bone reabsorption consists of three steps: 1. Adhesion of osteoclasts to bone matrix by podosomes 2. Matrix breakdown by H+ secretion 3. Enzymatic degradation of organic matrix Bone formation during embryo development is typically classified as: o Direct or intramembranous ossification (flat bones of skull, mandible, clavicle): pre-osteoblasts in the mesenchyme differentiate into osteoblasts and begin the synthesis and deposition of osteoid. Osteoid is the early non-mineralised matrix of the bone. The bone matrix develops into trabeculae. Some osteoblasts are trapped into the matrix and become osteocytes, the various ossification centres fuse to create spongy bone. The spaces between trabeculae are later filled with red bone marrow. o Indirect or endochondral ossification: most long bones and short bones are formed from a cartilage model. The embryonic cartilage is then replaced by bone tissue. The process consists in two fundamental events: formation of the hyaline cartilage, growing of the cartilage and replacement with bone tissue. 2020/2021 Isabella Ronchi Cartilage continually grows and is replaced with bone as soon as it grows. Bones lengthen entirely by growth of the epiphyseal plates. Cartilage cells form tall stacks 1. Chondroblasts in the upper stacks divide quickly 2. Epiphysis is pushed away from diaphysis 3. Lengthening of the entire long bone Older chondrocytes signal surrounding matrix to calcify, then die and disintegrate 1. Long trabecular of calcified cartilage are left on the diaphysis side 2. Osteoblasts cover trabeculae with bone tissue 3. Trabeculae are eaten away from their tips by osteoclasts Growing bones also widen as they lengthen. Bone is reabsorbed at the endosteal surface and added at the periosteal surface. Osteoblasts add bone tissue to the external surface of the diaphysis. Osteoclasts remove bone from the internal surface of the diaphysis. Bone remodelling consists in the continuous breakdown and reforming of bone tissue. The stress applied to bones during exercise are essential to maintain bone mass and bone strength. Bone is an active tissue: 5-7% of bone mass is recycled weekly. Spongy bone is replaced every 3-4 years and compact bone approximately every 10 years. Remodelling units deposit and reabsorb at perisoteal and endosteal surface. Bone deposition occurs always and more when the bone is injured or extra strength is needed. Remodelling requires a healthy diet (proteins, vitamins C, D and A and minerals). Bone reabsorption is accomplished by osteoclasts, through the secretion of lysosomal enzymes that digest organic matrix and HCl that converts calcium salts into soluble forms. Repair of fractures occurs in four steps: 1. Hematoma formation; mass of clotted blood 2. Callus formation: soft callus made of cartilage 3. Bony callus formation: hard callus made of spongy bone 4. Bone remodelling: removal of spongy bone and replacement with compact bone 2020/2021 Isabella Ronchi Blood circulates in a closed system of vessels. It has several functions; o Deliver oxygen and remove carbon dioxide and other metabolic wastes o Maintain temperature, pH (approx. 7.4, acidosis if 7.8) and fluid volume (4-5 L in women and 5-6 L in men) o Transport of nutrients absorbed in the intestine o Protection from blood loss (platelets) o Prevention of infections (antibodies and leukocytes) o Transport of hormones If we centrifugate a tube of blood we would get 45% formed elements at the bottom, a 1% buffy coat (leukocytes) in the middle and 55% plasma at the top. Plasma is made of 90% water and 8% proteins, which include albumin, Alpha and Beta Globulins, Gamma Globulins (antibodies) and fibrinogen. Most plasma proteins are produced by the liver, except for Gamma Globulins, which are produced by plasma cells. Albumin is responsible for the control of the oncotic pressure (osmotic pressure exerted by proteins). Globulins are involved in the transport of metals or other proteins. Fibrinogen and coagulation proteins are involved in blood coagulation mechanisms when blood exits from injured blood vessels. Plasma also contains organic nutrients (carbohydrates, amino acids, lipids and vitamins), hormones and metabolic waste (carbon dioxide and urea). The buffy coat includes leukocytes and platelets. The formed elements contain erythrocytes (red blood cells), leukocytes (white blood cells) and platelets (thrombocytes). Haematopoiesis means blood cell formation. It takes place in the bone marrow of the axial skeleton (medullary cavity of long bones, spaces of spongy bones). Haematopoietic stem cells give rise to all formed elements. When mature, new blood cells enter the sinusoids of the bone marrow and then the systemic circulation. Immature cells are kept inside the bone marrow by an endothelial barrier. During the embryological development haematopoiesis happens in the yolk sac in the first two months, then in the liver and the spleen and in the bone marrow from the 4th month. After birth it takes place only in the bone marrow: tibia and femur until 20-20 years of age and vertebrae, sternum and rib throughout the whole life, decreasing with age. There are two types of bone marrow: yellow bone marrow is inactive and has plenty adipocytes, red bone marrow is active. It’s made of a haematopoietic compartment and a stromal compartment, which protects the haematopoietic cells. The stromal compartment contains adipocytes, 2020/2021 Isabella Ronchi fibroblasts, endothelial cells, macrophages and blood vessels. There are three major cell populations in the bone marrow: o Self-renewing haematopoietic stem cells o Committed progenitor cells - Myeloblasts – myeloid progenitors - Lymphoblasts – lymphoid progenitors - Erythroblasts – erythroid progenitors o Maturing cells Balance between RBC production and destruction depends on an adequate supply of iron, amino acids and vitamin B. In addition, erythropoietin (EPO) is released by the kidneys in response to hypoxia. It causes a more rapid maturation of erythroid progenitors and increases circulating reticulocyte count in 1-2 days. Hypoxia is caused by a reduced RBC number (haemorrhage or increased RBC destruction), iron deficiency or reduced availability of oxygen (e.g., altitudes). Erythrocytes are around 5 million/mm3 in males and 4.5 million/mm3 in females. They have a life span of around 120 days in the bloodstream. They are removed in the spleen by macrophages and all important components (iron, amino acids) are recycled. They have a biconcave shape, with a diameter of about 7 µm. The nucleus and cytoplasmic organelles are lacking, because RBCs need space to transport oxygen and carbon dioxide. Sometimes during erythrocyte differentiation, the nucleus and the organelles aren’t lost completely, this type of cells are called reticulocytes and they make up 1% of total RBC count in a healthy individual. The membrane is made of 50% proteins, 40% lipids and 10% carbohydrates. Glycophorin is the integral membrane protein bearing carbohydrate chains for AB0 blood group. Blood type is based on the presence of 2 major antigens in RBC membranes: A and B. Another important blood group classification is based on the presence of the Rhesus (Rh) antigen on RBCs. A red blood cells is made of 66% water and 33% proteins, of which 95% is haemoglobin. Every erythrocyte contains 280 million molecules of haemoglobin. Hb has 2020/2021 Isabella Ronchi four chains of globin complexed to an iron-containing heme group for the binding of oxygen and carbon dioxide. Sickle-cell anemia is a mutation due to the substitution of the 287 amino acid in the Beta chain of the globin molecule. It’s found in 1/400 African Americans. Homozygosity is deadly, but in malaria infested countries, the heterozygous condition is beneficial. White blood cells are around 6000-10,000 per mm3. They have nuclei and do not contain haemoglobin. They can be divided into granular (neutrophils, eosinophils, basophils) or agranular (lymphocytes and monocytes) based on the staining of cytoplasmic granules. The general function is to fight invades by phagocytosis and long-term immune response. Leukocytes leave the bloodstream to reach the sites where their function is needed. Emigration (diapedesis) is mediated by specific adhesion receptors on leukocytes recognising counter-receptors on the endothelium. Leukocytes stick to and then squeeze between endothelial cells. Specific chemical signals (e.g., chemo-attractants) guide leukocytes to the precise site. NEUTROPHILS LYMPHOCYTES MONOCYTES EOSINOPHILS BASOPHILS 50-70% 20-40% 3-8% 2-4% 0.5-1% Diameter 10-14 Diameter 7-9 µm Diameter 14-18 Diameter 12-15 Diameter 12-15 µm µm µm µm Multi-lobed Round nucleus Oval or kidney- Bi or tri lobed Nucleus obscured nucleus (2-5 lobes) shaped nucleus nucleus Cytoplasm filled Thin layer of Abundant Acidophilic Basophilic granules with granules. basophilic, non- cytoplasm, granules, large (heparin and granular cytoplasm azurophilic ovoid granules histamine). Many granules with elongated lysosomes. crystalloid inside First wave at sites Proliferate and Migrate to Phagocytose Supplement mast of inflammation mature during the inflammatory sites antigen-antibody cell function at and defence immune response. and phagocytose complexes (high in inflammatory sites. against bacteria: B lymphocytes bacteria. Also allergy). Kill Bind IgE phagocytosis. (15%) produce perform trophic parasites. (hypersensitivity Peroxidases are antibodies and function for reactions) highly reactive recognise the tissues. substances in antigen. killing bacteria. T lymphocytes Dead neutrophils (80%) and Natural make up pus. Killer (5%) Survive 24h in the Can survive for Circulate for 3-4 Circulate in the circulation, 1-4 years maintaining days before blood for 24h, days in tissues. memory and can emigrating into survive in tissue for They die once their also re-enter the tissues where they several days supply of granules circulation. differentiate into has been macrophages (life exhausted. span few months) 2020/2021 Isabella Ronchi Platelets are 150,000-400,000 mm3 and they are about 2-5 µm in diameter. They derive from megakaryocytes that split into 2000-3000 fragments. Each fragment is enclosed in a piece of plasma membrane with many vesicles but no nucleus. The entire megakaryocyte cytoplasm is converted into a mass of proplatelets, which are released from the cell. The nucleus is extruded from the mass of proplatelets and individual platelets are released. Their life span in blood is about 10 days. They have a key role in homeostasis and coagulation. They form plugs to occlude the vascular damage by adhering to the exposed ECM at the margin of a wound. Later the platelet plug is reinforced by fibrin. They promote clot formation by providing a surface for the assembly of coagulation protein complex. They secrete factors that modulate the coagulation cascade and vascular repair. There are three types of granules: o Alpha granules: fibrinogen, platelet derived growth factor, other coagulation proteins o Lambda granules: lysosomal enzymes for removal of the clot o Delta granules: calcium, pyrophosphate, serotonin (vasoconstriction and platelet aggregation), ADP, ATP All immune cells come from precursors in the bone marrow and develop into mature cells through a series of changes that can occur in different parts of the body. Primary lymphoid organs: o Bone marrow – produces B cells and immature T cells o Thymus – instructs T cells Secondary lymphoid organs: o Lymph nodes – filter the lymph, site of activation of immune cells o Spleen – filters the content of the bloodstream o Lymphatic nodules o Diffused lymphoid tissue in the intestine and airways The overall function of the immune system is to prevent or limit infection by micro-organisms. An antigen is a non-self molecule or a self-modified molecule, able to engage and activate the receptors on the surface of T and B lymphocytes. Antigens are derived from a variety of sources including pathogens, host cells and allergens. Infectious microbes such as viruses and bacteria express another set of signals recognised by the immune system called pathogen-associated molecular patterns (PAMPs). The immune system can distinguish between normal healthy cells and unhealthy cells by recognising a variety of “danger” signals called danger-associated molecular patterns (DAMPs). When the immune system first recognises “danger” or “pathogen” derived signals, it responds to address the problem. If an immune response cannot be activated when there is sufficient need, infection arise. On the other hand, when an immune response is activated without a real threat or is not turned off once the danger passes, different problems arise, such as chronic inflammation, allergic reactions and autoimmune diseases. 2020/2021 Isabella Ronchi The immune system is complex and tightly regulated. There are numerous cell types that either circulate throughout the body or reside in a particular tissue. Each cell type plays a unique role, with different ways of recognising problems, communicating with other cells and performing their functions. The immune system is generally divided into innate and adaptive immunity. When cells of the innate immunity recognise Adaptive immunity takes longer (days), as it relies pathogens (or danger) a reaction occurs on the coordination and expansion of specific immediately (within minutes or hours). Cells of the adaptive immune cells. Adaptive immune cells are innate immunity include neutrophils, eosinophils, more specialised, with each B and T cell bearing basophils, mast cells, monocytes, macrophages, unique receptors that recognise specific epitopes dendritic cells and NK cells. Their main feature is of 1 antigen, rather than general patterns. the ability to respond quickly and broadly when a Antigens are captured and processed by innate problem arises. They have many receptors for immune cells and presented to T cells in the lymph different PAMPs and DAMPs. The response of nodes. When activated by an antigen, B cells make innate immunity is acute inflammation. If antibodies that neutralise pathogens. T cells carry inflammation is transient and not excessive, then out multiple functions, including killing infected it’s a physiological response. If it isn’t transient, it’s cells and activating other immune cells by always pathological. Innate immunity cells do not producing soluble mediators. T and B cells retain memory. maintain memory for their specific antigen, which protects the host to subsequent encounters with that specific pathogen. The thymus is a lymphoepithelial organ located in the superior mediastinum, above the heart. It differs from other lymphoid organs, because it arises from epithelial tissues, it doesn’t fight microbes and it doesn’t contain B cells. It has an epithelial capsule, a cortex and a medulla. It produces hormones (e.g., thymosin) to program lymphocytes. The cortex is made of close-packed cells, including epithelioreticular cells, thymocytes and macrophages. The medulla has less dense cells, including epithelioreticular cells, thymocytes, dendritic cells and macrophages. Type 1-3 epithelioreticular cells are in the cortex, whilst types 4-6 are in the medulla. The overall goal is to select thymocytes that will recognise only non-self antigens in the Major Histocompatibility Complex (a large locus containing genes that code for cell surface proteins) with their receptor. This selection brings to apoptosis of 96% of thymocytes. The blood-thymus barrier physically separates immature thymocytes from the bloodstream. This prevents unwanted penetration of antigens into the thymus, and it prevents immature thymocytes from entering the bloodstream. The barrier is made of three major elements: capillary endothelium + basal lamina + pericytes, perivascular connective tissue with macrophages and epithelioreticular cells. The thymus is very well developed in children. Involution begins after puberty: the thymus becomes smaller and is replaced by adipose tissue, but it retains some small functional area. Aged thymus presents Hassall corpuscles: type VI epithelial reticular cells displaying evidence of keratinisation. 2020/2021 Isabella Ronchi The lymphatic system is a one-way vessel system: from peripheral tissues to the heart. The lymph is the excess fluid that seeps out of the peripheral blood capillaries and remains in tissues. From tissues this liquid must return to the blood circulation. Lymphatic vessels convey fluid from the peripheral tissues to the veins. Capillary lymph vessels are permeable to proteins and cells. One-way mini valves allow fluid to enter but not to leave. Specialised lymphatic vessels located in the villi of the small intestine are called lacteals. Lymph nodes are located along lymphatic vessels and filter the lymph before it is returned to blood. Lymph nodes are a communication hub where immune cells sample information brought in from the body. Lymphocytes in the lymph node may recognise foreign antigens coming from a distant area. If this occurs, lymphocytes activate, replicate and then leave the lymph node to circulate and reach the site to attack the pathogen. There are different cells within lymph nodes: o Macrophage o Lymphocytes o Dendritic cells: present antigens to lymphocytes. Langerhans cells in the skin are dendritic cells: they take up antigens in the skin and migrate to the closest lymph nodes where they present antigens to T cells. o Reticular cells (produce reticular fibres) o Specialised high endothelial cells. Entry of lymphocytes in lymph nodes or exit of activated lymphocytes in peripheral tissues is mediated by particular adhesion receptors. Naïve lymphocytes enter lymph nodes via High Endothelial Venules (HEV), lined by cuboidal endothelial cells instead of squamous. High endothelial cells express specific carbohydrates that are recognised by receptors present only on the membrane of naïve lymphocytes. Lymphocytes can enter lymph nodes also via afferent lymphatics. Lymphocytes return to the blood via the thoracic duct. Chances of successful encounter are enhanced by re-circulation of lymphocytes (1-2% recirculate every hour). Cancer cells can detach from the primary tumour and enter lymph vessels. Analysis of sentinel lymph nodes helps understanding how a tumour spread. A sentinel lymph node is the first lymph node to which cancer cells are most likely to spread from the original site. Biopsy of sentinel lymph nodes is used to determine the extent or stage of cancer in the body. A negative result indicated that cancer has not spread to nearby lymph 2020/2021 Isabella Ronchi nodes or other organs. A positive result indicates that cancer is present in the sentinel lymph node and may be present in other nearby lymph nodes, and, possibly, in other organs. The spleen is located in the upper left quadrant of the abdominal cavity. Its main functions are: o Filter of the blood vascular system o Removal of blood-borne antigens in the white pulp o Removal and destruction of aged or defective blood cells in the red pulp o Reserve of blood volume o Haematopoiesis site in foetuses The white pulp (20%) is an accumulation of lymphocytes that surrounds branches of the splenic artery (periarterial lymphatic sheath). It’s important in immune response and it forms follicles of B and T lymphocytes with germinal centres also named Malpighian corspuscles. The red pulp (80%) is made of red pulp sinuses (large sinusoids) and red pulp cells (erythrocytes and macrophages). The diffused lymphatic tissue is made of: o Gut-associated lymphoid tissue (GALT) in the digestive tract o Bronchus associated lymphoid tissue (BALT) in the aiways o Mucosa associated lymphoid tissue (MALT) in the genital-urinary tracts They consist in lymphatic nodules wihtout a capsule and they’re located in the lamina propria, the loose connective layer below the epithelial lining. There are also lymphoid tissue nodules in the mouth, entrance to digestive and respiratory systems. They are specifically located in the Waldeyer ring (lingual tonsil, palatin tonsil and pharyngeal tonsil). The Peyer patches are lymphoid nodules in the GI tract: they resemble tonsils in structure and they capture and destroy bacteria in the intestine. The intestinal environment modulates cellular differentiation in the immune system to control defence against pathogens and tolerance to commensal species. Tolerance depends on appropriate innate immunity mechanisms that limit microbial entry to the intestinal tissues. Intestinal epithelial cells provide a physical barrier between the luminal micro-organisms and the underlying intestinal tissues to control homeostasis and tolerance. Specialised epithelial cells produce a mucus layer (Goblet cells) and secrete antimicrobial proteins (Paneth cells) that limit bacterial exposure to the epithelial cells.