Immunology Primer, Part A

Question 1

(a)    What protein did you first pick? Glycogen phosphorylase A (GPa), PDB# 1C8L, Synergistic Inhibition Of Glycogen Phosphorylase A By A Potential Antidiabetic Drug And Caffeine. http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=13355

(b)   How many different peptides did it have? GPa is a single peptide of 842 amino acids.

(c)    Did it have any beta-sheets? Yes. If so, how many? 25.

(d)   Did it have any alpha-helices? Yes. If so, how many? 34.

(e)    Did it have any hairpin loops? Yes. If so, how many? There appear to be 4.

(f)     Were there any other features worth noting? Yes. If so, what? GPa possesses a substrate binding site (for glycogen), and an allosteric site (for naturally occurring glucose-6-phosphate). The anti-diabetic drug, (-)(S)-3-isopropyl 4-(2-chlorophenyl)-1,4-dihydro-1-ethyl-2-methyl-pyridine-3,5, 6-tricarboxylate (Bay W1807) is a competitive inhibitor with AMP and is shown bound to the allosteric inhibitor site of this enzyme. Caffeine is also shown bound to a purine inhibitor site. These interactions have the potential to regulate glycogen metabolism. From http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&query_hl=1&list_uids=9384557: “W1807 binds at the GPb allosteric effector site, the site which binds AMP, glucose-6-phosphate and a number of other phosphorylated ligands, and induces conformational changes that are characteristic of those observed with the naturally occurring allosteric inhibitor, glucose-6-phosphate.” The structure of GP shown at http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=13355 illustrates this inhibitor bound to the allosteric site, as well as caffeine bound at the purine inhibitor site. Caffeine binds at that site by intercalating between two aromatic rings which stabilizes the T state conformation. The binding of W1807 to the allosteric site locks the enzyme into the T state, preventing phosphorylation of substrates. This structural model depicts in detail how it is an allosteric inhibitor that can prevent phosphorylation of glucose.

Question 2

(a)    What was the second protein you picked? Hemoglobin, Hb, PDB# 1VWT, T State Human Hemoglobin [alpha V96w], Alpha Aquomet, Beta Deoxy. http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=7599

(b)   How many different peptides did it have? Hemoglobin is a tetramer of 4 peptides – 2 alpha and 2 beta subunits. Subunits A and C have 141 residues and subunits B and D have 146 residues.

(c)    Did it have any beta-sheets? No. If so, how many? 0.

(d)   Did it have any alpha-helices? Yes. If so, how many? 7 in subunits A and C; 8 in subunits B and D.

(e)    Did it have any hairpin loops? No. If so, how many? 0.

(f)     Were there any other features worth noting? Yes. If so, what? Each subunit possesses a porphyrin heme group bound to iron, which in the R state would coordinate to oxygen.

(g)    In what ways was it similar to your first protein?  There is very little similarity, except for the fact that both have numerous alpha helices and are globular and compact.

(h)    In what ways was it different? Hb exists as a tetramer (4 chains) with alpha helices and GPa exists as a dimer of two identical chains side by side, consisting of beta sheets and alpha helices.

Question 3

(a)    What other sites did you visit in connection to one of your two selected proteins? I went to the abstract sites for the associated papers on glycogen phosphorylase at URLs:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&query_hl=1&list_uids=9384557, and http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&query_hl=1&list_uids=10548038, and http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&query_hl=1&list_uids=11368311. From there I could access the actual published papers on the last two URLs.

(b)   Briefly summarize the available information at two of these sites? These sites had abstracts describing how the anti-diabetic drug acts as an allosteric inhibitor for glycogen phosphorylase. The last site also discussed how caffeine can act as an inhibitor.

(c)    What new understanding did you gain from visiting these sites? That glycogen phorphorylase has more allosteric inhibitors (caffeine and the antidiabetic drug) than just previously known in current biochemistry texts.

Question 4

(a)    Protein 1QLF: Describe the structural characteristic of chains A and B. This protein consists of 2 structures (chains). Chain A (IQLF_a d1) is comprised of 9 beta sheets with 6 hairpin loops, disulfides bridges and 3 alpha helices in part of the chain; 9 beta sheet segments with 6 hairpin loops and disulfide bridges in a second part (IQLF_A d2). Chain B (1QLF_B d1) has 8 beta sheet segments with 6 hairpin loops and disulfide bridges.

(b)   What is a domain? Domains are defined in terms of structure and function of a protein. They often fold independently of other domains and are self stabilizing in the way they form the higher level structure in their shape. They are functionally unique in the biological role of which they function in the protein.

(c)    How many domains does each of these two chains have? Chain A has three, and chain B has one.

(d)   Identify what each chain is. 1QLF_A, chain A: Chain A has 276 amino acids. It is the MHC class 1 H-2DB heavy chain that has two domains that form the cleft (a1 and a2), and a Ig (a3) domain that forms one of the two stalks that connects to transmembrane segment (not shown). 1QLF_B, chain B: Chain B has 99 amino acids. It is the Immunoglobulin MHC-associated light chain. It is noncovalently associated with chain A. It is an Ig domain (designated b2-microglobulin) that acts as the other stalk section, but it does not connect to a transmembrane segment.

(e)    How did you find this information? By first going to http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6&db=t&Dopt=s&uid=11233 to view summary diagrams of the chains. Then by clicking on the ‘protein’ link at the left of each chain diagram to view text details of the chain. Then by clicking on ‘reports’ and dropping down the menu to ‘graphic’ to access secondary structure graphics. Also, by consulting the text book on MHC class 1 nomenclature, structure and function.

(f)     What is the function of each of these chains? Chain A forms the cleft to bind with a peptide antigen and also has a stalk portion that connects to a transmembrane element. The cleft portion has the three helix segments that form the cleft. Chain B is required for A chain to associate with so that class 1 molecules can be expressed on cell membranes.

(g)    What was the source organism of this protein? Chain A is from Mus musculus (house mouse), chain B is from Homo sapien, and chain C (polymer 3 antigen) is from the Sendai virus.

(h)    What is the molecular description of polymer 3? It is the peptide antigen that is bound to the cleft of the A chain – the glycopeptide K3g of the Sendai virus; It is designated 1QLF_C and has 9 amino acids.

Immunology Primer, Part B

Question 1

Intercellular collaborations resulting in cell signaling are a necessary part of any immune response. Cells need to interact with each other in specific ways, including direct contact through membrane-bound receptor-ligand interactions and/or soluble signal molecules, such as hormones and cytokines. Initial events in cell signaling lead to signal transduction, which as a term, includes events beginning with receipt of signal at the cell surface by whatever means, through intracellular signal pathways and second messengers, to affecting a response via gene activation and expressing.

a. Integrins

Integrins are an important group of adhesion molecules involved during cell migration and homing to specific tissues. Timothy Springer has been studying the cell signaling events associated with integrin binding and the specific molecular mechanisms involved.

(1)   Describe briefly the methods used in his laboratory to investigate the functional and structural aspects of integrins during adhesion events.

A starting step for investigation of integrin function and structure often involves preparation of soluble integrin heterodimers (Takagi et al. 2002). This soluble form differs from the in situ form in that the transmembrane portion has been removed so that the binding headpieces can be attached to an alternative peptide.  In the case of some studies (Takagi et al. 2001, Takagi wt al. 2003) soluble integrin heterodimers of integrin that bind to fibronectin are prepared with what is referred to as overlap extension polymerase chain reaction. In this procedure, the extracellular parts of wild type integrins such as a5 and b1 are fused together via a disulfide bridge that links the cysteines of segments of alpha-helical coiled coil peptides that were previously added to the dimer stalks. The disulfides are inserted mutational-wise. An insert is then made on the beta unit to extend its length. More significantly, the extension is a sequence of amino acids that can be cleaved by a protease called tobacco etch virus (TEV) protease. Later in the procedure, this site can be cleaved with TEV to simulate dissociation of the transmembrane or cytoplasmic domain portions of these soluble integrin subunits. This allows for the conformational change in the head end of the integrin dimer.

This approach also uses electron microscopy to obtain detailed 3D images of conformations in active and inactive states. This helps to determine if an integrin is in the extended (liganded) or bent conformation (unbound). Implementation of binding kinetics measurements help assess level of ligand binding in an assay.

Also used is ultracentrifugation sedimentation prior to electron microscopy to separate clasped (inactive) and unclasped (activated) forms of the mimetic soluble integrins because they fall through a gradient at different sedimentation rates (8.3S and 9.1S, respectively (Tagaki et el. 2001)). SDS-PAGE is used to separate protein fragments. Transfection techniques are used in cells to produce large quantities of the dimer as a result of expression from the cells. NMR is also used to deduce the chemical structure of parts of the integrin.

(2)   Summarize what has been discovered to date in terms of molecular conformational changes of integrins during adhesion and their role in cell signaling.

What has been found out about the above integrins and associated ligand, is that when the regions of the legs closest to the membrane separate, there is a conformational change in the head. This conformational change imparts a greater affinity of binding with a ligand and suggests that signaling is what is referred to as ‘inside-out’ – that is, intracellular signaling leads to the conformational change that increases the a5b1 integrin’s head binding potential for ligand.

Other studies have shown that the stalk areas (legs) of integrins are generally flexible and that this flexing is what allows, at least, the integrins to switch between inactive and active states (Takagi et al. 2002). Going from a bent to an extended conformation is what is associated with becoming active to bind a ligand. It is interesting to note that low electron density in the flexible region is affiliated with flexibility (Xiong et al. 2001).

The cytoskeleton actin-binding protein, talin, is thought to activate integrins. Talin binds the integrin’s cytoplasmic tail while in the active extended state (Calderwood 2004). Other studies have shown, based on NMR structure, that when integrins are bound and ligated that the epitope binding areas are exposed, and hidden in the bent conformation when inactive (Lu et al. 2001).

References

Shimaoka, M., Takagi, J. and Springer, T.A. 2002. Conformational regulation of integrin structure and function. Annual Review of Biophysics and Biomolecular Structure 31: 485-516.

Takagi, J., Erickson, H.P. and Springer, T.A. 2001. C-terminal opening mimics "inside-out" activation of integrin a5b1. Nature Structural Biology 8(5):412-416.

Takagi, J., Strokovich, K., Springer, T.A. and Walz, T. 2003. Structure of integrin a₅ß₁ in complex with fibronectin. The EMBO Journal 22(18):4607-4615.

b. Chemokine receptor signaling in B cell homing

Jason Cyster is interested in chemokines and receptor-ligand interactions involved in lymphocyte homing in splenic white pulp, peripheral lymph nodes, and Peyer’s patches.

(1)   Describe what is understood about chemokine receptor signaling in B cell homing.

There are a variety of chemokines that participate in recruiting inflammatory cells to prime them for activation in the immune response. Currently it is know that there are at least 40 members in this family (Cyster 2003). Among them are chemokines and their receptors that are involved in B-cell homing. Lymphoid chemokines, as they are called, include at least four: CXCL12/SDF1, CXCL13/BLC, CCL19/ELC, and CCL21/SLC (Cyster 2003). Their receptors are CXCR4, CXCR5, and CCR7 (in the case of the last two chemokines).

CXCL13 can be found in lymphoid follicles and body cavities. CXCL12 can be found in the epithelium, bone marrow, red pulp of the spleen, the medullary cord, and HEV (high endothelial venule). HEV is a specialized post-capillary venule with tall cuboidal endothelial cells that are found in lymph nodes and gut-associated lymphoid tissue. It is the site where lymphocytes recirculate from blood to lymph. CCL19 and CCL21 are found in the T zone, and CCL21 is additionally found in HEV.

B-cell migration into lymphoid follicles is facilitated by the chemokine, CXCL13 and its receptor, CXCR5 (Cyster 2003). B-cell entry into Peyer’s patches is facilitated by CXCR5 (Okada et al. 2002). It has been found that mice which lack the chemokine, CXCL13, B1 cells are in short supply in their peritoneal and pleural cavities. Those types of B-cells impart antibody capability to the body cavities (Ansel et al. 2002). Furthermore, B-cells express the CXCR5 receptor to the same extent as most other markers of differentiation which maximizes their ability to bind with the CXCL13 chemokine (Bowman et al. 2000).

What is known about how these chemokines and receptors work can be demonstrated by the mechanisms and events in the spleen and lymph nodes. Inside a follicle of the spleen, as a B-cell encounters antigen, it begins to express CCR7, moving to the T zone boundary with the follicle. The T zone is inundated with the SLC/CCL21 chemokines, which are the ligand for CCR7. Thus the migration towards and into the T zone occurs as a result of the affinity the chemokine and its receptor have for each other. Once inside the T zone, the B cells proliferate, some of them experiencing isotype switching, and many of the newly divided cells start to secret Ig. At this point the expression of both CCR7 and CXCR5 is reduced. Increased expression of CXCR4 occurs to facilitate homing of the B cells from the T zone to the red pulp, where the CXCR4 ligand, CXCL12 chemokine, occurs in high numbers. Thus, again, homing is accomplished by the detection of a ligand that effects an attraction through its receptor on the cell being attracted. Just how this is done is explained in the next question on chemotaxis. Finally, red pulp contains venous sinuses that allow passage of the plasma cells that originated from B cells. These cells can travel to places like the bone marrow where more CXCL12 also resides to attract them. A similar scheme of homing also exists in lymph nodes (Cyster 2003 and Reif et al. 2004).

References

Ansel, K.M., Harris, R.B.S. and Cyster, J.G. 2002. CXCL13 Is Required for B1 Cell Homing, Natural Antibody Production, and Body Cavity Immunity. Immunity 16:67-76

Bowman, E.P., Campbell, J.J., Soler, D., Dong, Z., Manlongat, N., Picarella, D., Hardy, R.R. and Butcher, E.C. 2000. Developmental Switches in Chemokine Response Profiles during B Cell Differentiation and Maturation. The Journal of Experimental Medicine 191(8):1303-1318.

Cyster, J.G. 2003. Homing of antibody secreting cells. Immunological Reviews 194(1): 48.

Gunn, M.D., Ngo, V.N., Ansel, K.M., Ekland, E.H., Cyster, J.G. and Williams, L.T. 1998. A B-cell-homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1. Nature 391: 799 – 803.

Okada, T., Ngo, V.N., Ekland, E.H., Förster, R., Lipp, M., Littman, D.R. and Cyster, J.G. 2002. Chemokine Requirements for B Cell Entry to Lymph Nodes and Peyer's Patches. The Journal of Experimental Medicine 196(1):65-75.

Reif. K., Okkenhaug, K., Sasaki, T., Penninger, J.M., Vanhaesebroeck, B. and Cyster, J.G. 2004. Cutting Edge: Differential Roles for Phosphoinositide 3-Kinases, p110g and p110d{delta}", in Lymphocyte Chemotaxis and Homing. The Journal of Immunology 173: 2236-2240.

c. Phosphoinositide 3-kinase involved in chemotaxis

Phosphoinositide 3-kinases are involved in chemotaxis of both neutrophils and lymphocytes.

(1)   Summarize what is understood to this point about the role and function of phosphoinositide 3-kinases.

Phosphoinositide 3-kinases are a group of molecules that exist within the cytosol of cells and that respond to the presence of chemokines via kicking off a cascade of signal transduction events inside the cell. Phosphoinositide 3-kinases respond to extracellular chemokine gradients in order to induce polarization of the cell. This polarization causes the cell to move up the chemokine gradient and it is the way in which chemotaxis of neutrophils and lymphocytes is accomplished to get them to the site of inflammation (Rickert et al. 2000).

Initially, the way this transduction works is via the presence of chemokines such as those involved in B-cell homing (e.g., CCL19 and CCL21) that bind with their receptors on the cell surface (Reif et al. 2004). These receptors are G protein-coupled receptors (GPCRs), and there are at least six involved in chemoattractant events associated with the immune response (Ward 2004). These receptors are among the ones described above in the section on B-cell homing.

Once the ligand binds to the receptor, a number of events occur within the cell that lead to its ability to migrate in the direction of the gradient. The first thing to happen is that the Gbg subunit of the G protein is released on the inner surface of the cell membrane. Once separated from this heterotrimeric G protein, the subunit activates phosphoinositide 3-kinase gamma (PI3Kg) and cytostolic tyrosine kinases (Rickert et al. 2000). PI3Kg is now able to catalyze the conversion of phosphatidylinolsitol 4,5-bisphosphate (PtdIns(4,5)P₂) to two products via phosphorylation: phosphatidylinolsitol 3,4,5-triphosphate (PtdIns(3,4,5)P₃) and phosphatidylinolsitol 3,4-bisphosphate (PtdIns(3,4)P₂) (Ward 2004).

As these products increase, specific GDP-GRP exchange factor (GEF) activity increase also occurs. GEF activates what is referred to as Rho GTPases. These then send signals to actin polymerization mechanisms (Rickert et al. 2000). It is here that the cell begins to change shape. As actin begins to polymerize at the leading edge of the cell, myosin II assembles at the rear of the cell, contributing to a forward driving force. The leading edge actin reorganization occurs close to the G protein-coupled receptors that released their Gbg subunit at that end of the cell. Such actin and myosin assemblies restructure the cell to ‘polarize’ to change its shape in such a way that the actin end of the cell moves toward the gradient of chemokines that initiated this cascade of signals (Stephens et al. 2002). Hence, now the cell is in a better ‘biomechanical’ shape and conformation to move towards the increased chemokine concentration through the epithelial layer during extravasation and subsequent migration. It should be noted that integrins and other adhesion molecules are involved in this process – that is to say, they are what had rolled the leukocyte into a position where it could then be swept into this chemokine gradient that exists from the endothelial layer to the deeper tissues or wherever the inflammation occurs.

References

Reif. K., Okkenhaug, K., Sasaki, T., Penninger, J.M., Vanhaesebroeck, B. and Cyster, J.G. 2004. Cutting Edge: Differential Roles for Phosphoinositide 3-Kinases, p110g and p110d, in Lymphocyte Chemotaxis and Homing. The Journal of Immunology 173: 2236-2240.

Rickert, P., Weiner, O.D., Wang, R., Bourne, H.R., Servant. G. 2000. Leukocytes navigate by compass: roles of PI3Kg and its lipid products. Trends in Cell Biology 10(11):466-473.

Stephens, L., Ellson, C. Hawkins, P. 2002. Roles of PI3Ks in leukocyte chemotaxis and phagocytosis. Current Opinion in Cell Biology 14(2):203-213.

Ward, S.G., 2004. Do phosphoinositide 3-kinases direct lymphocyte navigation? Trends in Immunology 25(2):67-74.

Question 3

There are many molecular mediators involved in host defense, both as innate responses to pathogenic challenges and in concert with adaptive immune responses. The following questions focus on only a few key areas of research.

a. TNF range of functions

Tumor necrosis factor [TNF] is a member of a large superfamily of proteins.

(1)   Summarize the range of functions found for members of the TNF superfamily.

There are 29 receptors and 19 ligands in this superfamily (Aggarwal 2003). This family has a number of beneficial roles as well as a number of negative roles. The positive biological functions include haematopoiesis, antibacterial, innate immunity, tumor regression, and immune surveillance. Abnormal or poorly regulated manifestations of members of this superfamily, on the other hand, include AIDS, Crohn’s disease, Alzheimer’s disease, multiple sclerosis, transplant rejection, type II diabetes, rheumatoid arthritis, heart failure, atherosclerosis, liver disease, allergic asthma, fever, tumor metastasis, tumorigenesis, pulmonary fibrosis, lymphoproliferative diseases, septic shock, osteoporosis, and systemic lupus (Aggarwal 2003). What follows is largely sourced from that reference.

TNF (tissue necrosis factor) a and b has been shown to possess limited capability to repress tumors. In fact, these two cytokines, known initially as lipopolysaccharide (LPS) and lymphotoxin (LT), were the first of the family to be discovered and extensively studied. It should be pointed out that ‘limited’ generally refers to the toxicity of TNF and that its use has to be balanced with suppressing its undesirable (systemic) toxic effect. The receptors for TNF-a and –b are TNFR1 and TNFR2, respectively.

TRAIL (TNF-related apoptosis-inducing ligand) kills tumor cells and does not harm other cells as does TNF. This, along with antibodies for its receptors, DR4 and DR5, are being studied extensively for therapeutic anti-oncogenic use.

TNF, TRAIL, and CD95L assist cytotoxic cells in the recognition and apoptosis of virus-infected cells. CD95 operates as a pro-apoptotic entity on other fronts however. It is thought that it is involved in clonal negative selection of T cells in the thymus. Indeed, a depletion of CD95/CD95L often leads to an autoimmune response characterized by uncontrolled proliferation of immune components.

As far as haematopoiesis, a number of the TNF members are involved in many roles. 4-1BBL stimulates CD8+ cells, CD27 and OX40 stimulate T cells, CD30L is affiliated with T helper 2 cell events, and CD40L is a required ligand in T cell dependent antibody functions. BAFF (B-cell activating factor) has receptors that are expressed by and essential to B cell maturation. Equally true, TNF and LT are associated with B cell proliferation. Dendritic cell development is dependent on CD40 and RANKL (receptor activator of NF-kb ligand). Interestingly, CD40 plays a major role in B cell maturation. CD40 will express on B cells and then bind to CD154 on T cells. This is a required step for release of cytokines that lead to B cell isotype switching (Guzman-Rojas et al. 2002). More on this is discussed in the next sub-part.

Finally, antimicrobial functions of members of the TNF superfamily are facilitated by TNF or TNFR1.

There are many more members of this superfamily than those mentioned above. But the range of biological roles discussed above help to outline the broad level of function of this superfamily.

(2)   Choose one of interest to you and describe the latest understanding of its mechanism of action and the specific roles ascribed to it.

TNF-a possesses two major functional roles: one that is apoptotic and the other which is anti-apoptotic. The anti-apoptotic pathway consists of activation of the NF-kb and AP-1 pathways. TNF-a exists as a trimeric form in solution outside the cell that can then bind to its transmembrane receptor. That receptor, TNFR1, possesses an intracellular death domain that is associated with the death domain of a protein called TRADD (TNF receptor-associated death domain). Once the receptor is activated, the SODD (silencer of death domain) protein is released from the cytosolic tail of the TNFR1 receptor so that TRADD can send its intracellular signal. [SODD prevents TRADD binding until TNF binds to the receptor – a control mechanism to prevent over stimulation of the TNF-affected pathways.] Acting through RIF, TRADD causes NIK (NF-kb inducing kinase) to assist phosphorylation by (IKK) Ikb-kinase of an inhibitor in a complex comprised of NF-kb and its inhibitor, Ikb. The inhibitor falls away and NF-kb can enter the nucleus to promote the transcription of genes that produce immune components. Alternatively, the apoptotic pathway involves TRADD signaling to two other mediating molecules, FADD (Fas-associated death domain) or RAIDD, which then cue procaspase to activate the caspase pathway which leads to apoptosis (Liz-Grana and Carnota 2001). Thus, the two major roles of TNF-a are proliferation of inflammatory elements or apoptosis of cells. Presumably, these latter cells are any cell that is infected or destined to die that expresses the receptors in tandem with signaling by other cells in an immune response.

References

Aggarwal, B.B. 2003. Signalling pathways of the TNF superfamily: a double-edged sword. Immunology 3:745-756.

Guzman-Rojas, L., Sims-Mourtada, J.C., Rangel, R. and & Martinez-Valdez, H. 2002. Life and death within germinal centres: a double-edged sword. Immunology 107(2):167-175.

Liz-Grana, M., and Carnota, J.J.G.R. 2001. Tumour necrosis factor. Genetics, cell action mechanism and involvement in inflammation. Allergol Immunol Clin 16:140-149.

b. CD40 role and mechanisms in cell death and generation of memory B cells

The receptors of TNF ligands and co-receptors also make up another superfamily of proteins. CD40 is one such protein and appears to be involved in apoptosis and in the development of memory B cells rather than plasma cells.

(1)   Describe what is known about CD40 and the mechanisms behind its involvement in cell death on one hand and generation of memory cells on the other.

The fate of B-cells has one of two outcomes: (1) positive selection that is associated with differentiation into memory cells with antigenic specificity, or (2) negative selection consisting of apoptosis (Martinex-Valdez et al. 1996). B-cells differentiate and become memory cells in the germinal centers of secondary lymphoid organs in a reaction called the GC (germinal center) reaction. Essentially, an immunoglobulin M (IgM)-expressing cell begins its journey to specificity as a naïve cell referred to in nomenclature as Bm1 or Bm2. This starts out in the extrafollicular portion of the lymphoid organ. Then they are exposed to antigen via antigen presenting cells such as follicular dentritic cells (FDC), undergoing gene rearrangement and somatic hypermutation. Eventually they will express CD40 which can then be bound to CD154 on a CD4+ T-cell (Siepmann et al. 2001 and Guzman-Rojas et al. 2002). The T cell then secretes cytokines and the B cell (now what is called Bm4-Ag-CD40) can undergo isotype switching where the constant region portions of antibody change isotypes. Next, the differentiated and mature B-cell can then migrate out of the germinal center (Guzman-Rojas et al. 2002). This is what is referred to as positive selection.

Those B-cells that do not bind to antigen or that are not matched well to Ag after somatic hypermutation will undergo apoptosis (Martinex-Valdez et al. 1996). This is facilitated by a surface protein called Fas (CD95) on the B-cell that binds with its receptor, FasL (CD95L), on the surface of a cytotoxic T-cell. This binding kicks off a signal cascade that leads to cell apoptosis. The Fas cytoplasmic tail associates with the FADD (Fas-associated death domain) protein in the cytosol. Collectively part of what is called the death-inducing signaling complex (DISC), they produce caspases (cysteine aspartate specific proteases) (Guzman-Rojas et al. 2002). It is these caspases that degrade the cell in apoptosis. This is then called negative selection.

B-cells automatically express Fas and possess the DISC complex, and are therefore pre-empted to apoptosis (Guzman-Rojas et al. 2002). Thus, binding of CD40 with its ligand can be viewed as a ‘rescue’ of the B-cell from cell death (Siepmann et al. 2001). The way this works inside the B cell is as follows: DISC is disabled by FLIP (FADD-like interleukin-1 converting enzyme inhibitory protein). FLIP expression is induced by CD40 signaling (van Eijk et al. 2001). In the end, the caspase cascade does not occur.

Guzman-Rojas et al. (2002) also note that self-determination can occur without the presence of the T-cell-CD154 receptor. Bm4-Ag-CD40-CD154 on the B-cell can also lead to memory/survival of the B cell. That is to say, a B-cell can express CD154 to bind to its CD40. The major problem with this of course is that defective B-cells can survive and contribute to cancer or autoimmune complications.

References

Guzman-Rojas, L., Sims-Mourtada, J.C., Rangel, R. and & Martinez-Valdez, H. 2002. Life and death within germinal centres: a double-edged sword. Immunology 107(2):167-175.

Martinez-Valdez, H., Guret, C., Bouteiller, O., Fugier, I., Banchereau, J., and Liu, Y. 1996. Human germinal center B cells express the apoptosis-inducing genes Fas, c-myc, P53, and Bax but not the survival gene bcl-2. J. Exp. Med. 183:971-977.

Siepmann, K., Skok, J., van Essen, D., Harnett, M. and Gray, D. 2001. Rewiring of CD40 is necessary for delivery of rescue signals to B cells in germinal centres and subsequent entry into the memory pool. Immunology 102:263–72.

Takahashi, T., Ohta, H. and Takemori, T. 2001. Fas Is Required for Clonal Selection in Germinal Centers and the Subsequent Establishment of the Memory B Cell Repertoire. Immunity 14 (2):181-192.

van Eijk, M., Defrance, T., Hennino, A., and de Groot, C. Death-receptor contribution to the germinal-center reaction. Trends Immunol 2001; 22:677–82.

c. Type 1 interferons

Type 1 interferons, IFN- a and IFN-b, are well known as antiviral mediators. They are also involved in other types of activity, including innate response to non-viral pathogens.

(1)   Summarize the recent understandings of their role in non-viral responses and the mechanisms of their functions.

Type 1 interferons are induced to engage in the immune response to non-viral pathogens such as bacteria, bacterial products, protozoa, and helminths. At least one known way that type 1 interferons operate is via the pattern recognition receptors of type 1 intereferons that are involved in a signal cascade similar to what happens when toll-like receptors encounter LPS (lipopolysacchardies) of bacteria (Bogdan et al. 2004). In fact, the toll-like receptors used include TLR-4. Other bacterial products like oligonucleotides and single stranded RNA exert a response as well. Oligonucleotides induct IFN-a through TLR-9.

The classical type 1 interferon pathway is the JAK/STAT (Janus kinase/signal transducers and activators of transcription). There are several that are activated such as STAT1, -2, -3, -4 , -5. All of these are part of the interferon pathway (Bogdan et al. 2004). In short, type 1 interferons help to induce phosphorylation of a Janus kinase, such as tyrosine kinase, and this results in genes that are transcribed in the nucleus (Kisseleva et al. 2002 and Ortmann et al. 200). These genes carry promoters consisting of IFN-stimulated response elements (ISRE). The gene products are mediators associated with the innate response. For example, infection by the protozoan, Leishmania, helps to produce IFN- a/b by macrophages. These interferons then activate the JAK/SAT pathway (STAT1 and -4) resulting in expression of iNOS (inducible nitric oxide synthase) and the killing of L. major (Bogdan et al. 2004).

The last reference cited includes a plethora of information also worth mentioning such as that it is now known that type 1 intereferons are likely produced by any cell. Also known is that there are 14 types of INF-a and one type of IFN-b. There are also other types of type 1 intereferons: IFN-w, IFN-k, IFN-e, and IFN-l. Not only is interferon production triggered by microbial organisms and their products, but also by mitogens and tumor cells. 

References

Bogdan, C., Mattner, J. and Schleicher, U. 2004. The role of type I interferons in non-viral infections. Immunological Reviews 202(1):33-48. 

Kisseleva, T., Bhattacharya, S., Braunstein, J., and Schindler, C.W. 2002. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 285(1-2):1-24.
 
Ortmann, R.A., Cheng, T., Visconti, R., Frucht, D.M., and O'Shea, J.J. 2000. Janus kinases and signal transducers and activators of transcription: their roles in cytokine signaling, development and immunoregulation. Arthritis Res. 2(1): 16–32.

d. Anti-microbial peptides

Anti-microbial peptides are a diverse group of small molecules found widely in nature, including outside the animal kingdom. They are significant in innate immunity.

(1)   Cite a specific example and describe its mode of action and potential for use as a therapeutic agent in controlling infectious disease.

Human b-defensin, hbd-1, is a naturally occurring 36 residue peptide that contains 3 disulfide bridges. The primary sequence is DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (Bauer et al. 2001). There are two types of hbd: 1 and 2 (Yang et al. 1999). As a b-defensin, it is expressed in epithelial cells of skin, kidneys, and trachea-bronchii of vertebrates. It is produced via innate signaling of the immune system when lipopolysaccharide from bacterium is detected by a cell (Yang et al. 1999). Like antimicrobial peptides in general, hbd-1 acts to disrupt bacterial membranes by lytic action, increased permeability or pore formation, all of which lyse the bacterium cell membrane (Bauer et al. 2001).

In addition to the above response associated with the innate system, it has also been shown that hbd-1 can act as a chemoattractant (Yang et al. 1999). Cells that are transfected and express the CCR6 chemokine receptor are homed to the site of invasion by this antimicrobial peptide. These cells are immature dentritic cells and memory T cells. As a result, hbd-1 interacts with elements of both the innate and adaptive immune system. And since the sequence of hbd-1 is known, as well as its three dimensional structure (Bauer et al. 2001), it can also be synthesized for use in therapeutic applications.

References

Bauer, F., Schweimer, K., Klüver, E., Conejo-Garcia, J.R., Forssmann, W.G., Rösch, R., Adermann, K. and Sticht, H. 2001. Structure determination of human and murine ß-defensins reveals structural conservation in the absence of significant sequence similarity. Protein Science (2001), 10:2470-2479. 

Yang, D., Chertov, O., Bykovskaia, S. N, Chen, Q,  Buffo, M.J., Shogan, J., Anderson, M.,  Schröder, J.M., Wang, J.M.,  Howard, O.M.Z., Oppenheim, J.J. 1999. b-defensins: Linking Innate and Adaptive Immunity Through Dendritic and T Cell CCR6. Science 286(5439):525-528.

My Question

Describe some of the ways that Rheumatoid Arthritis (RA) inflammation can be suppressed by blocking inflammatory cytokines, and provide an example of a drug that does this. Write a little on the inflammatory response to RA – what is happening in RA.

The causes of RA are not truly known. However, several pathway mechanisms are known that implicate cell-mediated response. The end result is an autoimmune response that activates a series of cytokine-mediated cell signaling processes and these processes lead to an increase in cells and proteins that degrade joint tissues. The overall result consists of a catabolic response that exceeds anabolism of replenishing matrix molecules. From a biochemical perspective, what is happening is the overproduction of inflammatory cytokines in the synovium.

Normal cartilage contains a type-II-collagen-fibril network with associated proteoglycans as well as proteoglycan aggregates that consist of a non-covalent association between aggrecan, hyaluronate, and linkage proteins. Complex carbohydrates are attached to cartilage proteoglycans. In this matrix are repeating disaccharide units of chondroitin and keratan sulfate, along with areas in which the chains are covalently bound to the core protein. The synovial membrane is one or two cells thick, with a sub-layer of loose connective tissue. Cells within the synovial lining include macrophages or fibroblasts. In early arthritis the synovial membrane perfuses into the cartilage. Synovial cell hyperplasia causes thickening. CD4+ T and B cells (plasma cells) then inundate the synovial membrane through a newly formed venule network. These cells and neutrophils exist in high concentrations in the fluid of the synovium (Choy and Panayi 2001). In full progression of the disease, the synovial membrane is characterized as a severe state of inflammation with immune cells destroying adjacent cartilage and bone (Mort and Billington 2001). The cytokine pathways involved in this joint destruction is thought to be mediated by TNF-a and interleukin-1 (Choy and Panayi 2001).

In summary: IL-1 and TNF- α induce migration of neutrophils into the synovial area. Neutrophils secrete proteases that breakdown proteoglycans. A reduction of proteoglycan attracts immune cells and molecules to perforate the matrix. This leads to an exposure of chondrocytes. The chondrocytes release metalloproteinases as a result of signaling by the two aforementioned cytokines. The metalloproteinases, stromelysin and collagenases, collectively reduce the connective-tissue matrix (Choy and Panayi 2001, Mort and Billington 2001). Proteolytic cleavage of the major components of the cartilage by proteases can be accomplished by four classes: serine/threonine proteases, cysteine proteases, aspartic proteases, and metalloproteases. Of these, metalloproteases are considered of primary importance. There are two families of metalloproteases: matrix metalloproteases (MMPs), which breakdown collagen and proteoglycan, and aggrecanases (ADAMTSs), which degrade aggrecan, a component of the proteoglycan aggregate (Mort and Billington 2001).

Treatment focuses on the inhibition of cytokines with the use of TNF- α and IL1 antagonists (Choy and Panayi 2001). Drugs include Infliximab. This is a chimeric (human and mouse) monoclonal antibody against TNF-α. It attaches to TNF-α so it can not attach to a receptor on the cell surface. A similar drug is Humira (Adalimumab). Another drug, Etanercept, consists of a soluble TNF-α type II receptor – IgG1 fusion protein. TNF-α too attaches to this drug so it can not attach to membrane receptors. Another way to block the cytokine receptor from binding with its inflammatory ligand is to bind a receptor antagonist or a monoclonal antibody to the receptor itself. Yet another approach that is being addressed is the use of SOCS (suppressor of cytokine signaling) proteins to bind to the JAK (Janus kinase) protein in the JAK-STAT (signal transduction activation of transcription) pathway. SOCS binds to JAK with high affinity to inhibit tyrosine kinase activity. Two types, SOCS-1 and -3, bind to the tyrosine in the activation loop of JAK through SH2 domains. This prevents access of the tyrosine kinase for phosphorylation. Inhibition of the activities of multiple cytokines occurs via SOCS blocking. That is, they target a shared cytokine signal transduction pathway (Ortmann et al. 2000, Rottapel 2001, Shouda et al. 2001, Egan et al. 2003, Ivashkiv and Tassiulas 2003). Normally, cytokines such as Interleukins and INFs attach to JAK-associated receptors on the cell surface to activate the JAK-STAT pathway. STATs then become phosphorylated, dimerize, and enter the nucleus. STATs attach to an enhancer region on DNA and act as transcription factors to initiate transcription of cytokine-dependent genes that produce proteins used by the immune system to mount a response. Normally, SOCS are expressed as negative regulators to block STAT activation. This approach merely exploits that natural mechanism (Kisseleva et al. 2002).

References

Choy, E.H.S. and Panayi, G.S.2001. Cytokine pathways and joint inflammation in
rheumatoid arthritis. N. Engl. J. Med 344:907-16.

Egan, P.J., Lawlor, K.E., Alexander, W.S. and Wicks, I.P. 2003. Suppressor of cytokine signaling-1 regulates acute inflammatory arthritis and T cell activation. J. Clin. Invest. 111:915-924.

Ivashkiv, L.B. and Tassiulas, I. 2003. Can SOCS make arthritis better? J. Clin. Invest. 111:795-797.

Kisseleva, T., Bhattacharya, S., Braunstein, J., and Schindler, C.W. 2002. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 285(1-2):1-24.

Mort, J.S. and Billington, C.J.. 2001. Articular cartilage and changes in Arthritis: Matrix degradation. Arthritis Res 3:337-341.

Ortmann, R.A., Cheng, T., Visconti, R., Frucht, D.M., and O'Shea, J.J. 2000. Janus kinases and signal transducers and activators of transcription: their roles in cytokine signaling, development and immunoregulation. Arthritis Res. 2(1): 16–32.

Rottapel, R.. 2001. Putting the brakes on arthritis: can suppressors of cytokine signaling (SOCS) suppress rheumatoid arthritis? J Clin Invest 108(12): 1745-1747.

Shouda, T., Yoshida, T., Hanada, T., Wakioka, T., Oishi, M., Miyoshi, K., Komiya, S., Kosai, K., Hanakawa, Y., Hashimoto, K., Nagata, K. and Yoshimura, A. 2001. Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis. J. Clin. Invest. 108:1781-1788.