Immunology and Cell Biology

We describe the generation of ovalbumin (OVA)‐specific, MHC class II‐restricted αβ T cell receptor (TCR) transgenic mice. Initial attempts at generating these transgenic mice utilized heterologous regulatory elements to drive the expression of cDNA genes encoding the separate α‐ and β‐chains of the TCR. Unexpectedly, T cells bearing the transgenic αβ TCR failed to emerge from the thymus in these mice, although the transgenes did modify endogenous TCR expression. However, subsequent modification of the approach which enabled expression of the TCR β‐chain under the control of its natural regulatory elements generated mice whose peripheral T cells expressed the transgenic TCR and were capable of antigen‐dependent proliferation. These results show that successful generation of MHC class II‐restricted, OVA‐specific αβTCR transgenic mice was dependent upon combining cDNA‐ and genomic DNA‐based constructs for expression of the respective α‐ and β‐chains of the TCR.

Allogeneic reactions have conventionally been considered as typical immune responses by one population of cells to antigens present on the other. This view is inadequate, since it does not explain many features of these reactions, among which are: (1) reactivity is much higher between different strains within a species than between species, in spite of the much greater antigenic disparity in the second case; (2) a very high proportion of cells may respond to allogeneic stimuli; (3) major histocompatibility differences are not essential for vigorous allogeneic reactions; (4) the responding population need not be immunologically competent to respond to antigens of the stimulating population; (5) the stimulating population must be both metabolically active and immunocompetent.

We have tried to produce a model of cell interaction which will account for these and other anomalies, while at the same time explaining both normal antigenic stimulation (through cell‐cell cooperation) and allogeneic interactions as examples of the same basic mechanism. The model is based on the Bretscher‐Cohn scheme of cell interaction. An allogeneic reaction is seen as having two stages: (1) Cells come together when antibody receptors on cells of one population combine with antigens on cells of the other. To this extent, our model is the same as the conventional one. It need not be the responding population which has the receptors, however. (2) A species‐specific proliferation signal passes between the cells. This is the same signal as is involved in normal antibody induction. Even antigen‐receptor bonds which are very weak may result in effective stimulation of one or both partners because of enhancing effect of this signal, and because the antigens involved are probably repeated over the cell surface, enabling multipoint binding. This explains the very high proportions of cells which proliferate. The exact outcome of any allogeneic interaction will depend on which of the two populations have antibody receptors for antigens on the other, which can produce the proliferative stimulus, and which can respond to either the proliferative signal alone or to this stimulus plus antigen.

Hyaluronan is a major component of synovial tissue and fluid as well as other soft connective tissues. It is a high‐Mr polysaccharide which forms entangled networks already at dilute concentrations (< 1 mg/mL) and endows its solutions with unique rheological properties. Physiological functions of hyaluronan (lubrication, water homeostasis. macromolecular filtering, exclusion, etc.) have been ascribed to the properties of these networks. Recently a number of specific interactions between hyaluronan and a group of proteins named hyaladherins have also pointed towards a role of hyaluronan in recognition and the regulation of cellular activities. Many more or less well documented hypotheses have been proposed for the function of hyaluronan in joints, for example, that it should lubricate, protect cartilage surfaces, scavenge free radicals and debris, keep the joint cavities open, form flow barriers in the synovium and prevent capillary growth.

Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the most versatile in terms of long‐term tracking of lymphocytes and their ability to quantify lymphocyte proliferation. They are the intracellular covalent coupling dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and the membrane inserting dye PKH26. Both dyes have the advantage that they can be used to track cell division, bothin vitroandin vivo, due to the progressive halving of the fluorescence intensity of the dyes in cells after each division. However, CFSE appears to have the edge over PKH26 based on homogeneity of lymphocyte staining and cost. Two other fluorescent dyes, although not suitable for lymphocyte proliferation studies, are valuable tracking dyes for short‐term (up to 3 day) lymphocyte migration experiments, namely the DNA‐binding dye Hoechst 33342 and the cytoplasmic dye calcein. In the future it is highly likely that additional fluorescent dyes, with different spectral properties to CFSE, will become available, as well as membrane inserting fluorescent dyes that more homogeneously label lymphocytes than PKH26.

Asthma is a chronic inflammatory disease of the airway that is characterized by cellular infiltration and activation. These processes are induced by overexpression of chemokines and cytokines, such as eotaxin, IL‐1β and GM‐CSF. These mediators are downstream targets for the transcription factors activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB), which control the expression of most immunomodulatory genes and whose activity and expression are elevated in asthma. Glucocorticoids are the most effective anti‐inflammatory drugs used in the treatment of chronic inflammatory diseases such as asthma. They act by binding to a specific glucocorticoid receptor (GR) that on activation translocates to the nucleus and either increases (transactivates) or decreases (transrepresses) the expression of responsive genes. Transrepression is the major mechanism of glucocorticoid action in inhibiting inflammatory gene expression. Thus, the ability of the transciption factors AP‐1 and NF‐κB to induce gene transcription is attenuated by GR. Although only 5–10% of asthmatic subjects are glucocorticoid‐insensitive, these subjects account for over 50% of the health‐care costs for asthma (> $6 billion per annum). Examining these patients also gives an insight into important aspects of glucocorticoid action in controlling inflammation and into the development of potential new drugs. Biochemical and genomic studies have indicated abnormal induction of the c‐Jun N‐terminal kinase (JNK) pathway in some of these patients. The ability of most patients to respond to dexamethasone with induction of histone acetylation correlated with nuclear translocation of GR. However, a subgroup of these patients had an inability to correctly interact with the basal transcription complex in spite of high levels of nuclear GR. This suggests that cross‐talk between pro‐ and anti‐inflammatory transcription factors may modulate activation of the transcriptional complex and thereby reduce steroid actions.

Real‐time quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) is the method of choice for rapid and reproducible measurements of cytokine or growth factor expression in small samples. Fluorescence detection methods for monitoring real‐time PCR include fluorogenic probes labelled with reporter and quencher dyes, such as Taqman probes or Molecular Beacons and the dsDNA‐binding dye SYBR Green I. Fluorogenic (Taqman) probes for a range of human and rat cytokines and growth factors were tested for sensitivity and compared with an assay for SYBR Green I quantification using real‐time fluorescence monitoring (PE Applied Biosystems Model 7700 sequence detector). SYBR Green I detection involved analysis of the melting temperature of the PCR product and measurement of fluorescence at the optimum temperature. Fluorogenic probes provided sensitive and reproducible detection of targets that ranged from low (<10 copies/reaction) to high (>10⁷ copies/ reaction) expression. SYBR Green I gave reproducible quantification when the target gene was expressed at moderate to high levels (≥1000 copies/reaction), but did not give consistently reproducible quantification when the target gene was expressed at low levels. Although optimization of melting temperature improved the specificity of SYBR Green I detection, in our hands it did not equal the reproducible sensitivity and specificity of fluorogenic probes. The latter method is the first choice for measurement of low‐level gene expression, although SYBR Green I is a simple and reproducible means to quantify genes that are expressed at moderate to high levels.

Engagement of the cell death surface receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation. Cell death mediated via Fas/FasL interaction is important for homeostasis of cells in the immune system and for maintaining immune‐privileged sites in the body. Killing via the Fas/FasL pathway also constitutes an important pathway of killing for cytotoxic T cells. Fas ligand is induced in activated T cells, resulting in activation‐induced cell death by the Fas/FasL pathway. Recently it has been shown that the Fas receptor can also be up‐regulated following a lesion to the cell, particularly that induced by DNA‐damaging agents. This can then result in killing of the cell by a Fas/FasL‐dependent pathway. Up‐regulation of Fas receptor following DNA damage appears to be p53 dependent.

Experimental visceral leishmaniasis (VL) caused by infection with Leishmania donovani results in the development of organ‐specific immunity in the two main target tissues of infection, the spleen and the liver. The liver is the site of an acute resolving infection associated with the development of inflammatory granulomas around infected Kupffer cells, and resistance to reinfection. Paradoxically, the spleen is an initial site for the generation of cell‐mediated immune responses, but ultimately becomes a site of parasite persistence with associated immunopathological changes. These include splenomegaly and a breakdown in tissue architecture that is postulated to contribute to the immunocompromized status of the host. The progressive development of splenic pathology is largely associated with high levels of TNF and interleukin (IL)‐10. Follicular dendritic cell (DC) networks are lost, whereas TNF mediates the destruction of marginal zone macrophages and gp38⁺ stromal cells, and IL‐10 promotes impaired DC migration into T‐cell areas with consequent ineffective T‐cell priming. Splenic stromal cell function is also altered, promoting the selective development of IL‐10‐producing DC with immunoregulatory properties. Ultimately, a fine immunological balance determines responses that effectively promote parasite clearance in the liver and those that promote pathology in the spleen, and future investigation aims to separate these responses to offer further means of parasite control in chronically infected VL patients.

In airways, mast cells lie adjacent to nerves, blood vessels and lymphatics, which highlights their pivotal importance in regulating allergic inflammatory processes. In asthma, mast cells are predominantly activated by IgE receptor cross linking. In response to activation, preformed mediators that are stored bound to proteoglycans, for example, TNF‐α, IL‐4, IL‐13, histamine, tryptase and chymase, are released. New synthesis of arachidonic acid metabolites (leukotriene C4 (LTC4), leukotriene B4 (LTB4) and prostaglandin D2 (PGD2)) and further cytokines is stimulated. Mediators from degranulating mast cells are critical to the pathology of the asthmatic lung. Mast cell proteases stimulate tissue remodelling, neuropeptide inactivation and enhanced mucus secretion. Histamine stimulates smooth muscle cell contraction, vasodilatation and increased venular permeability and further mucus secretion. Histamine induces IL‐16 production by CD8⁺ cells and airway epithelial cells; IL‐16 is an important early chemotactic factor for CD4⁺ lymphocytes. LTC4, LTB4 and PGD2 affect venular permeability and can regulate the activation of immune cells. The best characterized mast cell cytokine in asthmatic inflammation is TNF‐α, which induces adhesion molecules on endothelial cells and subsequent transmigration of inflammatory leucocytes. IL‐13 is critical to development of allergic asthma, although its mode of action is less clear.

The Toll/IL‐1 receptor (TIR) domain plays a central role in Toll‐like receptor (TLR) signalling. All TLRs contain a cytoplasmic TIR domain, which, upon activation, acts as a scaffold to recruit adaptor proteins. The adaptor proteins MyD88, Mal, TRIF, TRAM and SARM are also characterized by the presence of a TIR domain. MyD88, Mal, TRIF and TRAM associate with the TLRs via homophilic TIR domain interactions whereas SARM utilizes its TIR domain to negatively regulate TRIF. It is well established that the differential recruitment of adaptors to TLRs provides a significant amount of specificity to the TLR‐signalling pathways. Despite this, the TIR–TIR interface has not been well defined. However, structural studies have indicated the importance of TIR domain surfaces in mediating specific TIR–TIR interactions. Furthermore, recent findings regarding the regulation of adaptors provide further insight into the crucial role of the TIR domain in TLR signalling.