Molecular definition of the germinal centre stage of B-cell differentiation

Genomic-scale gene expression analysis provides views of biological processes as a whole that are difficult to obtain using traditional single-gene experimental approaches. In the case of differentiating systems, gene expression profiting can define a stage of differentiation by the characteristic expression of hundreds of genes. Using specialized DNA microarrays termed 'Lymphochips', gene expression during mature B-cell differentiation has been defined. Germinal centre B cells represent a stage of differentiation that can be defined by a gene expression signature that is not shared by other highly proliferative B-cell populations such as mitogenically activated peripheral blood B cells. The germinal centre gene expression signature is maintained to a significant degree in lymphoma cell lines derived from this stage of differentiation, demonstrating that this gene expression programme does not require ongoing interactions with other germinal centre cell types. Analysis of representative cDNA libraries prepared from resting and activated peripheral blood B cells, germinal centre centroblasts, centrocytes and tonsillar memory B cells has confirmed and extended the results of DNA microarray gene expression analysis.     

Production, characterization, and use of serpin antibodies

Serine protease inhibitors (serpins) constitute a still expanding superfamily of structural similar proteins, which are localized extracellularly and intracellularly. Serpins play a central role in the regulation of a wide variety of (patho) physiological processes including coagulation, fibrinolysis, inflammation, development, tumor invasion, and apoptosis. Serpins have a unique mechanism of inhibition that involves a profound change in conformational state upon interaction with their protease. This conformational change enables the production of monoclonal antibodies specific for native, complexed, and inactivated serpins. Antibodies, and assays based on these antibodies, have been helpful in elucidating the (patho) physiological function of serpins in the last decade. Serpin-specific antibodies can be used for: (1) structure-function studies such as detection of conformational changes; (2) identification of target-proteases; (3) the detection and quantification of serpin and serpin-protease complexes in bodily fluids by immunoassays such as ELISA, RIA or FACS; (4) detection of serpins in tissues by immunohistochemistry; and (5) possible therapeutical interventions. This review summarizes the techniques we have used to obtain and screen antibodies against extra- and intracellular serpins, as well as the use of these antibodies for some of the above-mentioned purposes.     

Physical characterization of serpin conformations

The native serpin fold is characterized by being metastable. This thermodynamic characteristic is manifested in the conversion of the native state to other more stable conformations. Whilst this structural transition is required for proteinase inhibition and regulation of a range of biological phenomena, inappropriate structural changes can result in a number of disease states. Identification of these alternative conformations has been essential in our understanding of serpin structure and function. However, identifying these alternative forms is also important if we are not to misinterpret data due to the formation of these states during in vitro studies. The different physical properties of these alternative serpin conformational states make it possible to use a range of standard laboratory techniques to identify these structures. In this chapter, we will outline these general approaches that can be used routinely to identify the alternative serpin conformational states.     

Identification and characterization of a serpin from Eimeria acervulina

Serpins are serine protease inhibitors that are widely distributed in metazoans but have not been previously characterized in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed and characterized. Random screening of an E.acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame (ORF) consisting of 1,245 nts that encode a peptide 413 amino acids in length with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues in length. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55 kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 kDa and 48 kDa in sporozoites (SZ) and merozoites (MZ) respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa which may correspond to secreted serpin. By immuno-staining the serpin was located in granules distributed throughout both the SZ and MZ but granules appeared to be concentrated in the parasite's anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis the endogenous serpin in MZ extracts did not form the expected high molecular weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes.     

Molecular gymnastics: serpin structure, folding and misfolding

The native state of serpins represents a long-lived intermediate or metastable structure on the serpin folding pathway. Upon interaction with a protease, the serpin trap is sprung and the molecule continues to fold into a more stable conformation. However, thermodynamic stability can also be achieved through alternative, unproductive folding pathways that result in the formation of inactive conformations. Our increasing understanding of the mechanism of protease inhibition and the dynamics of native serpin structures has begun to reveal how evolution has harnessed the actual process of protein folding (rather than the final folded outcome) to elegantly achieve function. The cost of using metastability for function, however, is an increased propensity for misfolding.     

Inhibitory mechanism of a cross-class serpin,the squamous cell carcinoma antigen 1

The squamous cell carcinoma antigen (SCCA) 1 and its homologous molecule, SCCA2, belong to the ovalbumin-serpin family. Although SCCA2 inhibits serine proteinases such as cathepsin G and mast cell chymase, SCCA1 targets cysteine proteinases such as cathepsin S, K, L, and papain. SCCA1 is therefore called a cross-class serpin. The inhibitory mechanism of the standard serpins is well characterized; those use a suicide substrate-like inhibitory mechanism during which an acyl-enzyme intermediate by a covalent bond is formed, and this complex is stable against hydrolysis. However, the inhibitory mechanism of cross-class serpins remains unresolved. In this article, we analyzed the inhibitory mechanism of SCCA1 on a cysteine proteinase, papain. SCCA1 interacted with papain at its reactive site loop, which was then cleaved, as the standard serpins. However, gel-filtration analyses showed that SCCA1 did not form a covalent complex with papain, in contrast to other serpins. Interaction with SCCA1 severely impaired the proteinase activity of papain, probably by inducing conformational change. The decreased, but still existing, proteinase activity of papain was completely inhibited by SCCA1 according to the suicide substrate-like inhibitory mechanism; however, papain recovered its proteinase activity with the compromised level, when all of intact SCCA1 was cleaved. These results suggest that the inhibitory mechanism of SCCA1 is unique among the serpin superfamily in that SCCA1 performs its inhibitory activity in two ways, contributing the suicide substrate-like mechanism without formation of a covalent complex and causing irreversible impairment of the catalytic activity of a proteinase.     

Regulatory T cells and control of the germinal centre response

Germinal centres (GCs) are specialised lymphoid microenvironments that form in secondary B-cell follicles upon exposure to T-dependent antigens. In the GC, clonal expansion, selection and differentiation of GC B cells result in the production of high-affinity plasma cells and memory B cells that provide protection against subsequent infection. The GC is carefully regulated to fulfil its critical role in defence against infection and to ensure that immunological tolerance is not broken in the process. The GC response can be controlled by a number of mechanisms, one of which is by forkhead box p3 expressing regulatory T (Treg) cells, a suppressive population of CD4⁺ T cells. A specialised subset of Treg cells – follicular regulatory T (Tfr) cells – form after immunisation and are able to access the GC, where they control the size and output of the response. Our knowledge of Treg cell control of the GC is expanding. In this review we will discuss recent advances in the field, with a particular emphasis on the differentiation and function of Tfr cells in the GC.     

A mathematical model for germinal centre kinetics and affinity maturation

We present a mathematical model which reproduces experimental data on the germinal centre (GC) kinetics of the primed primary immune response and on affinity maturation observed during the reaction. We show that antigen masking by antibodies which are produced by emerging plasma cells can drive affinity maturation and provide a feedback mechanism by which the reaction is stable against variations in the initial antigen amount over several orders of magnitude. This provides a possible answer to the long-standing question of the role of antigen reduction in driving affinity maturation. By comparing model predictions with experimental results, we propose that the selection probability of centrocytes and the recycling probability of selected centrocytes are not constant but vary during the GC reaction with respect to time. It is shown that the efficiency of affinity maturation is highest if clones with an affinity for the antigen well above the average affinity in the GC leave the GC for either the memory or plasma cell pool. It is further shown that termination of somatic hypermutation several days before the end of the germinal centre reaction is beneficial for affinity maturation. The impact on affinity maturation of simultaneous initiation of memory cell formation and somatic hypermutation vs. delayed initiation of memory cell formation is discussed.     

Molecular characterization of novel melanoma cell lines

We isolated two novel cell lines from different types of sporadic human malignant melanoma: the hmel1 line was obtained from a melanoma skin metastasis and the hmel9 cell line from a primary superficial spreading melanoma. The karyotype and pigmentation parameters were assessed in these cell lines. Cytogenetic analysis in early stages of culture revealed that both cell lines had chromosome instability and simultaneous growth of heteroploid subpopulations. The molecular analysis of some genes involved in melanoma showed that both cell lines harbor BRAF mutations. The unpigmented hmel1 and the pigmented hmel9 lines were found to express the tyrosinase gene. The tyrosine hydroxylase activity was detectable only in hmel9 cells and practically absent in the hmel1 cell line. This activity was found to be correlated with the relative tyrosinase protein amount in both melanoma cell lines. The biological behaviour in the two melanoma cell lines, derived from two different types of melanoma lesions displaying distinct clinical and histopathological features, confirms the heterogeneous characteristics of sporadic melanoma. Similarities and/or differences between cell lines extracted from different melanoma cases could be useful in the future for diagnostic, prognostic and therapeutic purposes.     

Molecular characterization of aromatase inhibitor-resistant,tamoxifen-resistant and LTEDaro cell lines

To determine potential genes involved in mediating resistance to aromatase inhibitors (AIs), a microarray study was performed using MCF-7aro (aromatase overexpressing) cells that are resistant to letrozole (T + LET R), anastrozole (T + ANA R) and exemestane (T + EXE R), as well as LTEDaro and tamoxifen-resistant (T + TAM R) lines for comparison. Based on hierarchical clustering, estrogen-responsive genes were found to be differentially expressed in AI-resistant lines versus LTEDaro and T + TAM R. Additional genome-wide analysis showed that gene expression profiles of the non-steroidal AI-resistant lines were most closely correlated and that T + EXE R lines exhibit differing profiles. Also, LTEDaro and T + TAM R lines are inherently different from expression profiles of AI-resistant lines. Further characterization of these resistant lines revealed that T + LET R, T + ANA R and LTEDaro cells contain a constitutively active estrogen receptor α (ERα) that does not require the ligand estrogen for activation. Ligand-independent activation of ERα does not activate identical estrogen-responsive gene profiles in AI-resistant lines as in LTEDaro lines, thereby establishing differing mechanisms of resistance. This ligand-independent activation of ER was not observed in the parental cell lines MCF-7aro, T + EXE R or T + TAM R cells. Based on the steroidal structure of EXE, our laboratory has shown that this AI has weak estrogen-like properties, and that EXE resistance involves an ER-dependent crosstalk with EGFR growth factor signaling. Recent studies in our laboratory pertaining to pre-clinical models of AI treatment revealed that intermittent use of EXE delays the onset of acquired resistance in comparison to continuous treatment. Specific molecular mechanisms involved in intermittent use of EXE are currently being explored, based on microarray gene expression profiling. Lastly, our laboratory has initiated a study of microRNAs and their potential role in regulating target genes involved in AI-resistance. Overall, we propose a model of acquired resistance that progresses from hormone-dependence (T + TAM R and T + EXE R) to hormone-independence (T + LET R and T + ANA R), eventually resulting in hormone-independence that does not rely on conventional ER signaling (LTEDaro).     

Serp-1, a viral anti-inflammatory serpin,regulates cellular serine proteinase and serpin responses to vascular injury

Complex DNA viruses have tapped into cellular serpin responses that act as key regulatory steps in coagulation and inflammatory cascades. Serp-1 is one such viral serpin that effectively protects virus-infected tissues from host inflammatory responses. When given as purified protein, Serp-1 markedly inhibits vascular monocyte invasion and plaque growth in animal models. We have investigated mechanisms of viral serpin inhibition of vascular inflammatory responses. In vascular injury models, Serp-1 altered early cellular plasminogen activator (tissue plasminogen activator), inhibitor (PAI-1), and receptor (urokinase-type plasminogen activator) expression (p < 0.01). Serp-1, but not a reactive center loop mutant, up-regulated PAI-1 serpin expression in human endothelial cells. Treatment of endothelial cells with antibody to urokinase-type plasminogen activator and vitronectin blocked Serp-1-induced changes. Significantly, Serp-1 blocked intimal hyperplasia (p < 0.0001) after aortic allograft transplant (p < 0.0001) in PAI-1-deficient mice. Serp-1 also blocked plaque growth after aortic isograft transplant and after wire-induced injury (p < 0.05) in PAI-1-deficient mice indicating that increase in PAI-1 expression is not required for Serp-1 to block vasculopathy development. Serp-1 did not inhibit plaque growth in uPAR-deficient mice after aortic allograft transplant. We conclude that the poxviral serpin, Serp-1, attenuates vascular inflammatory responses to injury through a pathway mediated by native uPA receptors and vitronectin.     

Axon growth and guidance genes identify T-dependent germinal centre B cells

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.     

Comparison of the effects of serpin A1, a recombinant serpin A1-IGF chimera and serpin A1 C-terminal peptide on wound healing

Serpin A1 (alpha1-antitrypsin, alpha1-proteinase inhibitor), a potent neutrophil elastase inhibitor, has therapeutic potential as a wound-healing agent. We compared the in vitro wound-healing action of serpin A1-IGF, a recombinant fusion protein of serpin A1(M351E-M358L) and insulin-like growth factor I with that observed in the presence of natural serpin A1 or A1-C26, the synthetic C-terminal 26 residue peptide of serpin A1, previously shown to have mitogenic and antiviral activities. All agents reduced wound sizes in monolayers of the kidney epithelial cell line LLC-PK1 and in primary cultures of human skin fibroblasts. Wound reduction in primary human keratinocytes was only observed with the serpin A1-IGF chimera. None of the factors stimulated cell proliferation using a colorimetric assay, with the exception of the serpin A1-IGF chimera, which caused a significant increase of cell proliferation and thymidine incorporation in human skin fibroblasts. However, wound healing by the A1-IGF chimera was reduced in keratinocytes in the presence of mitomycin C, suggesting a role of cell proliferation in wound reduction. The hydrophobic A1-C26 peptide significantly increased the production of collagen I in skin fibroblasts, an appealing asset for skin care applications.     

Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium

Endosialin, the antigen identified with monoclonal antibody FB5, is a highly restricted 165-kDa cell surface glycoprotein expressed by tumor blood vessel endothelium in a broad range of human cancers but not detected in blood vessels or other cell types in many normal tissues. Functional analysis of endosialin has been hampered by a lack of information about its molecular structure. In this study, we describe the purification and partial amino acid sequencing of endosialin, leading to the cloning of a full-length cDNA with an open reading frame of 2274 base pairs. The endosialin cDNA encodes a type I membrane protein of 757 amino acids with a predicted molecular mass of 80.9 kDa. The sequence matches with an expressed sequence tag of unknown function in public data bases, named TEM1, which was independently linked to tumor endothelium by serial analysis of gene expression profiling. Bioinformatic evaluation classifies endosialin as a C-type lectin-like protein, composed of a signal leader peptide, five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats), followed by a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. Carbohydrate analysis shows that the endosialin core protein carries abundantly sialylated, O-linked oligosaccharides and is sensitive to O-sialoglycoprotein endopeptidase, placing it in the group of sialomucin-like molecules. The N-terminal 360 amino acids of endosialin show homology to thrombomodulin, a receptor involved in regulating blood coagulation, and to complement receptor C1qRp. This structural kinship may indicate a function for endosialin as a tumor endothelial receptor for as yet unknown ligands, a notion now amenable to molecular investigation.