Balancing thymocyte adhesion and motility: a functional linkage between beta1 integrins and the motility receptor RHAMM.

Thymocyte differentiation involves several processes that occur in different anatomic sites within the thymus. Therefore, thymocytes must have the ability to respond to signals received from stromal cells and adopt either adhesive or motile behavior. We will discuss our data indicating human thymocytes use alpha4beta1 integrin, alpha5beta1 integrin and RHAMM to mediate these activities. Immature multinegative (MN; CD3-4-8-19-) thymocytes use alpha4beta1 and alpha5beta1 integrins to mediate weak and strong adhesion. This subset also uses alpha4beta1 integrin to mediate motility. As thymocytes differentiate, they begin to express and use RHAMM to mediate motility in conjunction with alpha4beta1 and alpha5beta1 integrins. Motile thymocytes use beta1 integrins to maintain weakly adhesive contacts with substrate to provide traction for locomoting cells, thus weak adhesion is a requirement of motile behavior. Hyaluronan (HA) is also required by thymocytes to mediate motility. HA binding to cell surface RHAMM redistributes intracellular RHAMM to the cell surface where it functions to mediate motility. We propose that the decision to maintain adhesive or motile behavior is based on the balance between low and high avidity binding conformations of beta1 integrins on thymocytes and that RHAMM:HA interactions decrease high avidity binding conformations of integrins pushing the balance toward motile behavior.

Beta1-integrins control spontaneous adhesion and motility of human progenitor thymocytes and regulate differentiation-dependent expression of the receptor for hyaluronan-mediated motility.

The functions of the receptor for hyaluronan-mediated motility (RHAMM) and beta1-integrin in adhesion and motility were analysed for human progenitor multinegative (CD3- 4- 8- 19-) thymocytes (MN Thy). Both alpha4beta1- and alpha5beta1-integrins are expressed by MN Thy, but only alpha4beta1 mediates fibronectin (FN)-dependent adhesion and motility. Freshly isolated MN Thy lack expression of RHAMM and their motility is RHAMM independent. Prolonged surface expression of RHAMM on MN Thy is dependent upon FN. RHAMM expression, which occurs prior to surface expression of CD3/T-cell receptor (TCR), was found to be inhibited by cross-linking of alpha4-, alpha5- and beta1-integrins, as was the prolonged FN-dependent phase of RHAMM expression. To confirm that RHAMM expression had been down-regulated rather than rendered cryptic by treatment with immobilized anti-integrin monoclonal antibody (MoAb), RHAMM mRNA levels were analysed. Transcription of RHAMM was decreased 7-12-fold by treatment with immobilized anti-alpha4 or anti-alpha5, and twofold by anti-beta1. Prior to expression of CD3/TCR and RHAMM, alpha4beta1 regulates migratory behaviour. After MN Thy differentiate to acquire CD3/TCR in vitro or in vivo, their motility becomes dependent upon both RHAMM and beta1-integrins. Integrins play a direct role in FN-dependent, RHAMM-independent motility of MN Thy, and an indirect role in RHAMM-dependent motility. This work shows that beta1-integrins are primary mediators and regulators of fundamental cell behaviours required during migratory phases of T-cell differentiation that occur prior to the expression of CD3/TCR.

Epitope mapping of monoclonal antibodies specific for the directly cross-linked mesodiaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacterium Pectinatus cerevisiiphilus.

Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells. All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding. Mab reactivity with purified PG confirmed this. Epitope mapping revealed the Mabs in total recognize four binding sites on the PG. Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells. No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria. These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria. These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.

Brewing spoilage Lactobacilli detected using monoclonal antibodies to bacterial surface antigens.

A panel of thirteen monoclonal antibodies (Mabs) was assembled that reacts with surface antigens on eight of eleven Lactobacillus brewing spoilage organisms, including one or more of L. brevis, L. buchneri, L. casei-alactosus, L. plantarum, or unspeciated isolate(s). Immunoblotting was done to identify the antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment and by surface antigen extraction of washed bacteria. Protease susceptibility of extracted surface antigens was also examined. In most cases, Lactobacillus surface antigens detected by the Mabs appear to be noncovalently bound proteins readily altered or removed from the bacterium by various environmental conditions. This research identifies brewing conditions that need to be tested to ascertain whether bacterial surface antigen-reactive Mabs can be used for the rapid, sensitive, and specific detection of Lactobacillus brewing spoilage organisms.

During human thymic development, beta 1 integrins regulate adhesion, motility, and the outcome of RHAMM/hyaluronan engagement.

During human thymic differentiation, interactions between fibronectin (Fn)/beta1 integrins and hyaluronan (HA)/RHAMM control motility and Fn/beta1 integrins mediate spontaneous Fn-dependent adhesion. Multinegative (MN, CD3-4-8-) thymocytes exhibit strong spontaneous adherence to Fn (75%) that was efficiently inhibited by anti-alpha5beta1 and only weakly inhibited by anti-alpha4beta1. The relatively weak adherence of unfractionated thymocytes to Fn required both alpha4beta1 and alpha5beta1. Video time-lapse microscopy indicates that a subset of thymocytes also undergo spontaneous Fn-dependent motility mediated by alpha5beta1, alpha4beta1, and the HA-receptor RHAMM, but not by CD44. The loss of motility after hyaluronidase treatment of thymocytes indicated that motility is strongly dependent on HA. Of motile cells, 55% were DP, 19% were DN, and 24% were CD4+SP, but only 1% were CD8+SP. Overall, for MN thymocytes, beta1 integrin mediated Fn-adhesion, but after expression of CD4/CD8, beta1 integrins mediated Fn-dependent motility. Treatment with the activating anti-beta1 mAb QE.2E5 inhibited thymic motility and converted otherwise nonadherent thymocytes to an adherent state. High-avidity interactions via integrins appear to supercede the motogenicity of RHAMM and HA, suggesting that integrin avidity may regulate RHAMM. During thymic development, changes in adhesion or motility appear to be mediated by integrin avidity modulation.