Search

During the late-phase asthmatic response, eosinophils migrate to the bronchial tissue and cause severe damage. In this study we compared in vivo primed eosinophils from patients with allergic asthma with eosinophils from healthy control subjects in their adhesion behavior to tumor necrosis factor-alpha-activated endothelium under flow conditions (0.8 dyn/cm2). More eosinophils from patients with asthma adhered to activated endothelium, compared with cells from healthy control subjects (1,237 +/- 126 versus 887 +/- 94 cells/mm2, respectively). In the presence of blocking antibodies directed against very late antigen-4 and E-selectin, the residual binding of the cells of individuals with allergic asthma was significantly higher than that of the healthy control subjects (353 +/- 64 versus 123 +/- 31 cells/mm2, respectively, P < 0.02). In addition, secondary tethering or formation of clusters of the eosinophils of patients with allergic asthma was significantly increased compared with the healthy control subjects (cluster indices 1.8 +/- 0.3 versus 0.8 +/- 0.2, respectively, P < 0.05). Because patient cells showed an enhanced interaction with platelets during the perfusions, the role of P-selectin on platelets was investigated. Blocking antibodies directed against P-selectin reduced the enhanced binding and clustering of eosinophils of patients with allergic asthma. We conclude that P-selectin-bearing platelets contribute to secondary tethering processes of eosinophils to activated endothelium. Therefore, platelets might play an important role in the chronic inflammatory processes of these patients.

Cytotoxic T-lymphocyte (CTL) epitopes on the 19-kDa lipoprotein from Mycobacterium tuberculosis were identified by the use of lipopeptides and their cytokine profile studied. Selection of candidate CTL epitopes was based on synthetic peptides derived from the amino acid sequence of the 19-kDa lipoprotein showing major histocompatibility complex class I (MHC-I) binding motifs (H-2D(b) and H-2L(d)). Their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S was studied. Similar studies were performed with peptides, in which the anchor amino acid of the H-2D(b) MHC-I motif was replaced by alanine. Three out of five peptides with H-2D(b) or H-2Ld binding motifs and their corresponding lipopeptides as well, up-regulated and stabilized the H-2D(b) molecules on RMA-S cells. Replacement of the anchor amino acid residues of the H-2D(b) MHC-I motif by alanine revealed that the anchor amino acid asparagine at position 5, contributed more to binding of peptide to H-2D(b) molecules than leucine at position 11. The closely related lipopeptides LP19c and LP19d, in combination with incomplete Freund's adjuvant (IFA), induced CTL responses in C57BL/6 (H-2(b)) mice. These CTLs could recognize the naturally processed antigen, i.e. the 19-kDa antigen protein produced and processed by the EX-19 cell line. The capacity of the various lipopeptides to induce CTL correlated well with the ability of the (lipo)peptide to up-regulate and to stabilize H-2D(b) molecules. Lipopeptide LP19c primed spleen cells showed a T helper type one profile after in vitro stimulation with P19c and P19d 19 kDa peptides. The approach to characterize presumptive 19-kDa CTL epitopes might lead to selection of promising CTL epitopes, which can be applied in the development of subunit tuberculosis vaccines. (C) 2000 Elsevier Science Ltd. All rights reserved.

Eosinophils exhibit a rolling interaction with E-selectin-expressing endothelium, and need to be activated by inflammatory mediators to firmly adhere to this surface. This study shows that IL-8 induces a transient arrest of unprimed eosinophils that roll on E-selectin present on TNF-α-activated HUVEC in an in vitro flow chamber. This process was antagonized by neutralizing Abs directed against IL-8 showing the specificity of the IL-8 effect. Furthermore, blocking Abs against both α4 and β2 integrins inhibited the IL-8-induced transient arrest while these Abs had no effect when they were added separately. The IL-8-induced arrest was pertussis toxin sensitive. Studying the effect of IL-8 in more detail, we evaluated putative changes in intracellular Ca2+ concentration in eosinophils induced by IL-8. We could show that IL-8 induces a transient rise in intracellular Ca2+ concentration in ∼40% of the cells provided that the eosinophils are interacting with endothelial cells or fibronectin-coated surfaces. Together these data show that resting eosinophils respond to IL-8 provided that the cells adhere on physiological surfaces. The induction of a transient arrest provides a new level of chemokine-induced regulation of leukocyte adhesion under flow conditions.

Mycobacterium tuberculosis is a facultatively intracellular bacterium that causes tuberculosis (TB). Between 1985 and 1991, TB increased 33% in Switzerland, 30% in Denmark, 20% in Norway, and 18% in both Ireland and the United States. Presently, TB affects 1.7 billion people worldwide. With 55 million cases of active disease, there are 8 million new cases and around 3 million deaths per year (2, 14). This situation, mainly in the developing countries, is directly related to a “weakness” of the immune system caused by human immunodeficiency virus infection, cancer, and the application of immunosuppressive drugs in chemotherapy (6). The situation is further complicated by the emergence of multi-drug-resistant strains (2, 6). Mycobacterium bovis BCG is the currently used vaccine against tuberculosis, but its efficacy varies widely, from 0 to 90% (15). BCG mainly activates CD4+ T cells, but it fails to activate CD8+ T cells (14). Cytotoxicity mediated by CD4+ cells, however, is probably not sufficient to eradicate the mycobacterium, which has an excellent capacity to survive intracellularly. Several studies have demonstrated the importance of CD8+ cytotoxic T lymphocytes (CTLs) against M. tuberculosis infection in mouse (CD8 knockout) models (11) and humans (12, 19, 22). However, only a few studies have described the mycobacterial antigens and the epitopes recognized by CD8+ CTLs (4, 31, 32, 38). Induction of cellular immunity, covering both CD4+ and CD8+ T-cell activation, may therefore be required for providing protection against M. tuberculosis (13). For the identification of CTL epitopes on viral, bacterial, and tumoral proteins, different strategies can be followed. One approach used is immunization with the whole protein to generate CTL responses or CTL clones and the subsequent mapping of the epitope by overlapping synthetic peptides (23). An alternative approach is to select 8- to 11-amino-acid (aa)-long peptides based on known major histocompatibility complex class I (MHC-I) binding motifs. These peptides are then studied for their ability to up-regulate and stabilize MHC-I expression (44). The advantage of this approach is that it limits the number of peptides to be synthesized and to be tested in animal experiments. The 38-kDa lipoglycoprotein, one of the better-studied antigens of M. tuberculosis, is actively secreted but is also partly attached to the surface of mycobacteria by a lipid tail (43). The protein induces strong antibody and T-cell responses and provides partial protection against M. tuberculosis infection in mice when it is administered either entrapped in biodegradable microparticles or in the form of a DNA vaccine (37, 46). Epitopes on the 38-kDa lipoglycoprotein have been recognized by CD4+ T cells and studied extensively with both mice (34, 36) and humans (35, 42). Recently, a number of epitopes recognized by CD8+ T cells (CTL epitopes), with an H-2b restriction, have also been described (38, 46, 47). This report describes the identification of CTL epitopes with an H-2Db restriction by synthesizing peptides with MHC-I binding motifs and studying their ability to up-regulate and stabilize MHC-I molecules. Positive peptides in these assays do not necessarily induce cytotoxic T-cell responses, even when they are injected with incomplete Freund’s adjuvant (IFA) (3, 9, 10, 38). The immunogenicity of these peptides, however, can be strongly improved by attachment of lipid tails, preferably with a spacer amino acid sequence between peptide and lipid (5, 18, 20, 26, 33). Previously, it has been demonstrated that the cysteine-serine-serine (CSS) spacer amino acid sequence was required for the efficient induction of CTLs against malaria epitopes (33). Therefore, palmitic acid with a CSS spacer amino acid sequence was linked to the amino terminus of the peptide, and mice were immunized with these lipopeptides in combination with IFA. Two new H-2Db CTL epitopes were identified on the 38-kDa mycobacterial lipoglycoprotein, which increases our knowledge for future M. tuberculosis vaccine design. The relationship between the ability to up-regulate and stabilize MHC-I molecules and the capability to induce CTLs by peptides and lipopeptides will be discussed.