Differential production of and migratory response to beta chemokines by human microglia and astrocytes

Heparin-binding EGF-like growth factor (HB-EGF) is a mitogen for smooth muscle cells (SMC) and is detected in SMC and macrophages in atherosclerotic plaques, suggesting that HB-EGF may be associated with the pathogenesis of atherosclerosis. The present study indicates that cilostazol, a phosphodiesterase III inhibitor, suppresses the expression of HB-EGF in rat aortic SMC and in U-937 cells, a macrophage-like cell line, stimulated by lipopolysaccharide. Further, cilostazol diminished the induction of HB-EGF mRNA by methylglyoxsal, which is a reactive dicarbonyl metabolite produced as the result of a glycation reaction and which might be associated with macroangiopathy caused by hyperglycemia. Cilostazol suppressed the production of HB-EGF protein in the conditioned medium of SMC. These data suggest that cilostazol might act by suppressing the progression of atherogenesis by means of suppressing the expression of HB-EGF in SMC and macrophages.     

Differential binding of chemokines to glycosaminoglycan subpopulations

BACKGROUND: Specificity in leukocyte trafficking is likely to depend on sequential interactions between various cell-type-specific leukocyte adhesion molecules, such as selectins and integrin ligands, and leukocyte-activating factors. A major class of leukocyte-activating factors, the chemokines, are soluble polypeptides that bind glycosaminoglycans, the polysaccharide components of cell-surface and extracellular-matrix proteoglycans. It has been suggested that cell-surface glycosaminoglycans of the heparin/heparan sulfate class mediate the presentation of chemokines to leukocytes by vascular endothelial cells. We investigated the possibility that specificity exists in the recognition of particular heparin/heparan sulfate structures by chemokines, by studying the binding of four members of the chemokine superfamily to heparin and heparan sulfate. RESULTS: Using affinity co-electrophoresis we found that interleukin-8 preferentially bound a subfraction of heparin that also showed increased affinity for melanoma growth stimulating activity (also known as MGSA, GRO or GRO alpha). This same subfraction of heparin, however, was not significantly preferentially bound by platelet factor 4 or neutrophil activating factor-2. Subsequent analysis of the three-dimensional structures of these chemokines indicated that their ability to discriminate among heparin subspecies correlates with the presence of paired glutamic acid residues within the putative glycosaminoglycan-binding site of the chemokine. This observation led to predictions about the relative affinities of heparan sulfate for interleukin-8 and platelet factor 4, predictions that were confirmed by further binding assays. CONCLUSION: Chemokines can bind selectively to subsets of heparin/heparan sulfate glycosaminoglycans, raising the possibility that glycosaminoglycans participate in determining the specificity of leukocyte recruitment in vivo.     

A model mechanism for the chemotactic response of endothelial cells to tumour angiogenesis factor

In order to accomplish the transition from avascular to vascular growth, solid tumours secrete a diffusible substance known as tumour angiogenesis factor (TAF) into the surrounding tissue. Endothelial cells which form the lining of neighbouring blood vessels respond to this chemotactic stimulus in a well-ordered sequence of events consisting, at minimum, of a degradation of their basement membrane, migration, and proliferation. A model mechanism is presented which includes the diffusion of the TAF into the surrounding host tissue and the response of the endothelial cells to the chemotactic stimulus. The model accounts for the main observed events associated with the endothelial cells during the process of angiogenesis (i.e. cell migration and proliferation); the numerical results compare very well with experimental observations. The situation where the tumour (i.e. the source of TAF) is removed and the vessels recede is also considered.     

Differential chemotactic activities of sensory neuropeptides for human peripheral blood mononuclear cells

We studied the chemotactic effects of calcitonin gene-related peptide, vasoactive intestinal peptide, substance P (SP), and secretoneurin on PBMC and PBL using micropore filter assays. All four peptides induced migration of PBMC, whereas only calcitonin gene-related peptide, vasoactive intestinal peptide, and SP were chemotactic for PBL. Secretoneurin, known to induce monocyte chemotaxis, was unable to affect lymphocyte migration. Effects of SP on PBL were characterized by checkerboard analyses and represented true chemotaxis. Both T and B cells responded chemotactically to SP, the functional activity of SP residing in its C-terminal amino acid sequence. Involvement of neurokinin (NK) receptors was supported by inhibition of SP-induced migration of PBL with an NK1 receptor antagonist and induction of migration with [Sar9, Met(O2)11]SP and [PyrGlu6, Pro9]SP(6-11), two specific agonists for NK1 receptors, but not with [beta-Ala8]NK A(4-10), an agonist for NK2 receptors. PBL chemotaxis to SP was abolished by inhibition of tyrosin kinase but not by that of protein kinase C. Preincubation of PBL with pertussis or cholera toxin inhibited SP chemotaxis, indicating that in PBL, NK receptors for chemotaxis probably are coupled with G protein and involve a tyrosin kinase signaling pathway. We conclude that, together with calcitonin gene-related peptide and vasoactive intestinal peptide, SP is a lymphocyte chemoattractant, whereas secretoneurin, which is coreleased from sensory nerve endings, is not.     

Differential antigen recognition by T cell populations from strains of mice developing polar forms of granulomatous inflammation in response to eggs of Schistosoma mansoni

In humans, infection with schistosome helminths can lead to dissimilar forms of clinical disease. Likewise, in the experimental mouse system, identical infection protocols with Schistosoma mansoni cause a more severe granulomatous disease in the C3H strain than in the C57BL/6 strain. To address this difference, we developed panels of schistosomal egg antigen (SEA)-specific T cell hybridomas to compare the responses of C3H and C57BL/6 mice to the major egg antigen p40. All derived C3H T cell hybridomas, despite being clonally distinct and restricted by either I-Ak or I-Ek, responded to recombinant fragment 15-1 of the p40 antigen, while none of the C57BL/6 T cell hybridomas did. Consistent with the observed monoclonal T cell responses, polyclonal lymph node cells from schistosome-infected C3H mice reacted strongly to fragment 15-1, which contrasted sharply with the weak response displayed by the C57BL/6 strain. Moreover, studies with congenic mice demonstrated that the strong CD4+ T cell response to fragment 15-1 was under major histocompatibility complex control and segregated with the H-2k haplotype. These findings suggest that a dominant T cell response against a major egg antigen may represent a risk factor for the development of severe disease.     

Immunoreactivity of neural crest-derived cells in thymic tissue developing under the rat kidney capsule

In order to study the functional development of a thymus in an experimental model, small pieces of adult rat thymic tissue were cultured for 9 days and implanted under the kidney capsule of littermates. The tissues were examined with a panel of antibodies raised against thymic and neural factors and neural crest cells at intervals from 5 to 13 days. At 5 days post-implantation, there were groups of L1+ cells within the implants that reacted with antibodies raised against neural and neural crest cell markers. L1+ cells were highly mitotic, rounded cells measuring 8.7 +/- 0.6 micrometer in diameter. Double immunostaining with different combinations of antibodies showed that 94% of the L1+ cells were also TH+, and many were HNK-1/NCAM+, PGP 9.5+, NGF+, chromogranin A+, VIP+, S100+, CGRP+, GAD+, and A2B5+. A few were also pan-cytokeratin+. These results indicate that these cells are derived from neural crest derived cells and belong to the neuroepithelial line of development. The L1+ cells were most numerous before nerves appeared (about Day 9) and reduced in number and extent as the thymus differentiated. The neural crest cells occasionally had long cytoplasmic extensions, but it was not possible to decide if they formed the nerves that appeared in the implants. Adult thymuses also contained a population of L1+ and HNK-1/NCAM+ cells, mainly in the subcapsular cortex, the septa, and the medulla. These cells could be a source of neural crest cells able to repopulate the implant. The adult thymus may always contain a reservoir of cells potentially capable of producing neuropeptides and transmitter factors required for thymic growth and regeneration.     

Microbial parasites versus developing T cells: an evolutionary 'arms race' with implications for the timing of thymic involution and HIV pathogenesis

The thymus attempts to ensure that T cells which emerge from it are able to discriminate self from nonself. As such, it is a potential 'backdoor' through which microbial parasites can enter, manipulate the host into perceiving them as 'self', and thereby avoid immune surveillance. It is proposed that the host has evolved to overcome this parasitic strategy by rapidly producing large numbers of long-lived T cells very early in life (closing the backdoor), before the developing individual has significant contact with infectious organisms, and while still under the protection of its mother's intact immune system. Hence the capacity of the thymus to function efficiently early in the lifespan would have been strongly favored by natural selection. It is well established in evolutionary biology that strong selection favoring enhanced early function easily accommodates, through pleiotropy, the accumulation of later occurring negative effects, and it is through this process that thymic involution and subsequent immune system senescence may have evolved. Once a large pool of competent T cells has been produced, even those microbes capable of contaminating the thymus usually can be eliminated, or at least contained. However, microbes that both destroy peripheral T cells (particularly peripheral T cells that are activated against them), and contaminate the thymus (leading to deletion of potential replacements of the destroyed peripheral cells), may be able to eventually overcome the immune system, thus producing disease after a long period of apparent latency. Human immunodeficiency virus, which is initially well controlled by the immune system, may become unleashed via this process.     

Clonal deletion of V beta 5+ T cells by transgenic I-E restricted to thymic medullary epithelium

A variety of cell types expressing MHC class II molecules is known to function as APC in vitro. We employed the Ig kappa gene enhancer and promoter to target the class II E alpha gene, and thereby I-E, exclusively to B cells to address their APC function in vivo. Although transgenic I-E was expressed on B lymphocytes, we unexpectedly obtained I-E on thymic medullary epithelium but not macrophages and at low frequency on dendritic cells. Using these transgenic mice, we constructed bone marrow irradiation chimeras with I-E expressed only on medullary epithelium, in order to determine the role of this cell type in tolerance by clonal deletion in the thymus. Although it is accepted that bm-derived cells play a primary role in deletion, and thymic epithelium can delete clones to a lesser degree, the role of cortical vs medullary thymic epithelium has not been directly dissected. We demonstrate that medullary epithelium alone can tolerize by partial deletion of I-E-reactive V beta 5+ T cells. These results indicate a role for medullary epithelium in deletion during the later stages of thymic development, and support the notion that positive and negative selection of developing T cells can occur in distinct temporal and anatomic compartments.     

T cell receptor alpha gene rearrangement and transcription in adult thymic gamma delta cells

T cells belong to two separate lineages based on surface expression of alpha beta or gamma delta T cell receptors (TCR). Since during thymus development TCR beta, gamma, and delta genes rearrange before alpha genes, and gamma delta cells appear earlier than alpha beta cells, it has been assumed that gamma delta cells are devoid of TCR alpha rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic gamma delta cells undergo VJ alpha rearrangements more frequently than immature alpha beta lineage thymic precursors. Sequence analysis shows VJ alpha rearrangements in gamma delta cells to be mostly (70%) nonproductive. Furthermore, VJ alpha rearrangements in gamma delta cells are transcribed normally and, as shown by analysis of TCR beta-/- mice, occur independently of productive VDJ beta rearrangements. These data are interpreted in the context of a model in which precursors of alpha beta and gamma delta cells differ in their ability to express a functional pre-TCR complex.     

Differential regulation of in vitro cytokine production by human blood cells in response to methylmethacrylate

The effect of methylmethacrylate (MMA) on human whole blood cultures (WBC) obtained from healthy donors was investigated. Lymphocyte transformation and cytokine production, that is, interleukin 6 (IL-6), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha), were used to evaluate the immunological activities of MMA. Primary cytotoxicity testing of MMA in Jurkat cells showed that this compound decreased the cell proliferation to 50% at a concentration of >60 mmol/L. Similarly, MMA significantly decreased lymphocyte transformation in either phytohemagglutinin (PHA) or Staphylococcus aureus protein A (SpA) activated WBC at 100 mmol/L. In contrast to activated WBC, MMA had no observed effect on resting blood cells. Cytokine expression in WBC seemed differentially modulated by MMA. There was a tendency for IL-6 production in both resting and PHA-stimulated WBC to be upregulated, while IL-6 induced in SpA stimulated cultures was downregulated. TNF-alpha was slightly increased by MMA in resting WBC at early incubation periods, and it was slightly downregulated in response to PHA or SpA activation. Suppression of IFN-gamma secretion was observed in WBC with or without PHA or SpA stimulation. The overall results demonstrated that MMA at physiological levels could influence the cytokine production in normal human blood cells in vitro. Alterations of cytokine production patterns by MMA indicate that this compound has multiple regulatory effects on immune reactions in normal human blood.     

Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments

In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho-stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cell-specific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4-4, TB4-20, and TB4-28. While TB4-4 and TB4-20 are both epithelium specific, TB4-28 detects an epitope expressed on both epithelial- and mesenchymal-derived stromal cells. TB4-4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4-20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.     

Mouse dendritic epidermal T cells exhibit chemotactic migration toward PAM 212 keratinocyte culture supernatants

Dendritic epidermal T cells (DETCs) are Thy-1+, CD45+, CD3+, CD4-, CD8-, and T-cell receptor-V gamma 3/V delta 1+ leukocytes that reside normally in adult mouse skin. We have demonstrated previously that keratinocytes serve as adhesion substrates for DETCs, and that interleukin 7 (IL-7), which is produced by keratinocytes, serves as a growth factor for DETCs. The present study was conducted to address the mechanisms by which DETCs migrate into the epidermis, reasoning that keratinocytes may also be a source of chemotactic activity. Short-term DETC lines were 35S-labeled and tested for migration toward Pam 212 keratinocyte culture supernatants using a modified Boyden chamber method; cell movement from upper chambers toward test samples in lower chambers was traced by counting radioactivity. DETC displayed rapid (within 60 min) and marked (> 50%) migration toward keratinocyte supernatants. The majority of cells that had migrated into keratinocyte supernatants expressed the V gamma 3 T-cell receptor, thus verifying that the migrating cells were DETCs. Addition of keratinocyte supernatants to the upper chambers completely blocked migration, suggesting its chemotactic nature. By contrast, no DETC migration was observed toward 3T3 fibroblast supernatants. Chemotactic activities were 1) produced by Pam 212 cells even in the absence of serum; 2) greater than 12 kD in size; 3) heat and pH labile; 4) trypsin sensitive; and 5) precipitated by 60-100% ammonium sulfate. Several cytokines (e.g., IL-1 alpha and IL-8) failed to mediate DETC migration when added to the lower chambers. Likewise, the same cytokines, when added to the upper chambers, failed to inhibit DETC migration toward Pam 212 supernatants. These results support our hypothesis that keratinocytes facilitate the residence of DETC in epidermis by secreting unique chemotactic factors, by providing adhesion substrates, and by elaborating specific growth factors.     

Differential TCR signaling and the generation of memory T cells

PURPOSE: Serum soluble interleukin-2 receptor level is a sensitive and quantitative marker of lymphocyte activation. We determined levels of serum soluble interleukin-2 receptor in children with reflux nephropathy to evaluate its clinical significance in the prediction for the progression of renal injuries. MATERIALS AND METHODS: Serum soluble interleukin-2 receptor values were determined in 63 children with reflux nephropathy. The group consisted of 37 boys and 26 girls 10 to 18 years old. T cells (naive and memory), B cells and macrophages were evaluated immunohistochemically in the scarred kidneys of 4 other patients (3 boys and 1 girl 5 to 16 years old) who underwent nephrectomy due to severe reflux nephropathy with little function seen on (99m)technetium-dimercapto-succinic acid (DMSA) renal scan. Levels of serum soluble interleukin-2 receptor were measured by an enzyme-linked immunosorbent assay. We simultaneously determined serum levels of creatinine and beta2-microglobulin, and urinary levels of alpha1-microglobulin and microalbumin. Individual functions of the right and left kidneys were estimated by renal dimercaptosuccinic acid uptake. RESULTS: Levels of serum soluble interleukin-2 receptor in the patients who had low total uptake of DMSA (right uptake plus left uptake) were significantly higher than those from patients with normal total uptake. Levels of serum soluble interleukin-2 receptor correlated significantly with levels of creatinine (r=0.616, p <0.0001) and beta2-microglobulin (r=0.803, p <0.0001), and levels of urinary alpha1-microglobulin (r=0.753, p <0.0001) and microalbumin (r=0.673, p <0.0001). A significant negative correlation was observed between levels of serum soluble interleukin-2 receptor and total DMSA uptake values (right uptake plus left uptake r=-0.678, p <0.0001). In the scarred kidneys leukocyte infiltrates were markedly increased in fibrosed spaces. The predominant cell type in these lesions was memory T cells. CONCLUSIONS: These results suggest that elevated levels of serum soluble interleukin-2 receptor are likely to reflect activated T cells in the kidneys of patients with reflux nephropathy and may be a useful predictor of progression of renal injury in these children.     

Development and evolutionary aspects of thymic T cell education to neuroendocrine self

Thymic epithelial cells, including nurse cells (TECs/TNCs), from various species synthesize neuroendocrine-related precursors belonging to neurohypophysial, tachykinin and insulin hormone families. The thymic repertoire of neuroendocrine-related polypeptides illustrates at the molecular level the paradoxical role of the thymus in both T cell positive and negative selection. On the one hand, these precursors are a source of signals which interact with neuroendocrine-type receptors expressed by target pre-T cells according to the cryptocrine type of cell-to-cell signaling. On the other hand, the same precursors constitute a source of self-antigens which are presented to pre-T cells by the thymic major histocompatibility complex system. Basically, the model of thymic T cell education to neuroendocrine self was established by the identification in TECs/TNCs of immunoreactive (ir) oxytocin as the self-antigen of the neurohypophysial family. Nevertheless, through the expression in TECs/TNCs of ir-neurokinin A and ir-insulin-like growth factor-II, the model also applies to the tachykinin and insulin hormone families.     

The central role of chemokines (chemotactic cytokines) in the immunopathogenesis of ulcerative colitis and Crohn''s disease

Rats with large thalamic lesions affecting the mediodorsal (MDn) and intralaminar (ILn) nuclei are impaired performing delayed matching to sample tasks (DMTS). To determine the neurological basis of this deficit, we trained rats to perform a place DMTS task and then compared the effects of excitotoxic lesions of the MDn, the ILn, and the lateral internal medullary lamina (L-IML). The MDn lesion had little effect. The ILn group was significantly impaired throughout 8 months of training. The L-IML group exhibited an intermediate level of impairment. Varying the sample response requirement, retention intervals, and trial-to-trial congruence in the side reinforced, had predicted effects, as did variations in response latency. However, none of these factors interacted with the treatment effects. These findings indicate that DMTS performance is disrupted by lesions of the ILn but not the MDn.