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Regulatory T cells (Tregs) were shown to be involved into the pathogenesis of acute rejection after transplantation. The suppressive activity of the total regulatory T cell pool depends on its percentage of highly suppressive HLA-DR(+) -Treg cells. Therefore, both the suppressive activity of the total Treg pool and the extent of HLA-DR expression of HLA-DR(+) -Tregs (MFI HLA-DR) were estimated in non transplanted volunteers, patients with end-stage renal failure (ESRF), healthy renal transplant patients with suspicion on rejection, due to sole histological Bord-R or sole acute renal failure (ARF), and patients with clinically relevant borderline rejection (Bord-R and ARF). Compared to patients with only Bord-R or only ARF, the suppressive activity of the total Treg cell pool was exclusively reduced in patients with clinically relevant Bord-R. In parallel, the HLA-DR MFI of the DR(+) -Treg subset was significantly decreased in these patients, due to a significantly lower proportion of DR(high+) -Tregs, which were shown to have the highest suppressive capacity within the total Treg pool. Our findings clearly demonstrate that the determination of the HLA-DR MFI of the HLA-DR(+) -Treg subset allows a highly sensitive, specific and non-invasive discrimination between patients with clinically relevant Bord-R (Bord and ARF) and patients with subclinical rejection or other causes of transplant failure.

Methylprednisolone is widely used to improve immune suppression in transplanted patients threatened by acute rejection. Recently, we showed that the suppressive activity of a Treg cell population depends decisively on their percentage of highly suppressive HLA-DR(high+)-Treg cells, which are strongly reduced in rejecting transplant patients. In order to examine whether the composition of the total CD4(+)CD127(low+/-)FoxP3(+)-Treg cell pool with different Treg-subsets (DR(high+)CD45RA(-)-Tregs, DR(low+)CD45RA(-)-Tregs, DR(-)CD45RA(-)-Tregs, DR(-)CD45RA(+)-Tregs) is affected by methylprednisolone bolus therapy we compared the percentages of these four different Treg cell subsets in transplant patients with biopsy proven rejection before and after steroid bolus therapy (n=23). In patients treated with steroid bolus therapy, the percentage of the naïve DR(-)CD45RA(+)-Tregs was significantly decreased, whereas the percentage of the DR(+)CD45RA(-)-Tregs was significantly increased. By that, the strongest increase was detected for the most suppressive DR(high+)CD45RA(-)-Tregs. However, these effects were only temporarily and closely associated to the duration of the bolus therapy. Our results suggest that besides various anti-inflammatory effects on cells of the adaptive and innate immune system, methylprednisolone also has the capacity to enhance the suppressive activity of the total Treg cell pool by increasing its percentage of highly differentiated and highly suppressive DR(high+)CD45RA(-)-Tregs.

Renal injury is accompanied by the presence of infiltrating inflammatory cells in the glomerulus and tubulointerstitium. FTY720 modifies lymphocyte migration into injured tissues by lymphocyte sequestration to secondary lymphoid organs. The purpose of this study was to examine the potential of FTY720 to influence the inflammatory response in a nonimmunological model of renal failure. Sham-operated and [Formula: see text] nephrectomized (SNX) Sprague-Dawley rats received two different doses of FTY720 or vehicle orally for 14 wk. Treatment with FTY720 reduced glomerular and tubulointerstitial damage in SNX rats but failed to stabilize creatinine clearance. The increase in gene expression of chemokine receptors CCR1, CCR2, and CCR5 in kidneys of vehicle-treated SNX rats was significantly attenuated by high-dose FTY720. Treatment with high-dose FTY720 tended to normalize RANTES and MCP-1 renal gene expression. FTY720 affected not only glomerular and tubulointerstitial lymphocytes, but M1 and M2 phenotype macrophages were also reduced. FTY720 significantly reduced key mediators of renal inflammation and fibrosis. FTY720 also decreased immunoregulation of M2 macrophages, which are beneficial for tissue remodeling and repair.

SummaryPersistent, localized Staphylococcus aureus infections, refractory to antibiotic treatment, can result in massive tissue destruction and surgical intervention is often the only therapeutic option. In that context, we investigated patients with S. aureus-induced infection at various sites, apparent as either olecranon bursitis, empyema of the knee joint or soft tissue abscess formation. As expected, a prominent leucocyte infiltrate was found, consisting predominantly of polymorphonuclear neutrophils (PMN) (up to 75%) and to a lesser extent of T lymphocytes and natural killer (NK) cells. In line with their bactericidal capacity, PMN expressed the high-affinity receptor for IgG, CD64 and the lipopolysaccharide (LPS) receptor CD14; moreover, the oxygen radical production in response to the bacterial peptide f-MLP was enhanced, while chemotactic activity was greatly reduced. The more intriguing finding, however, was that a portion of PMN had acquired major histocompatibility complex (MHC) class II antigens and CD83, indicative of a transdifferentiation of PMN to cells with dendritic-like characteristics. Of note is that a similar transdifferentiation can be induced in PMN in vitro, e.g. by gamma interferon or by tumour necrosis factor alpha. Co-cultivation of transdifferentiated PMN with autologous T lymphocytes resulted in prominent T cell proliferation, provided that S. aureus enterotoxin A was added. Taken together, persistent S. aureus infection induces PMN to acquire characteristics of dendritic cells, which in turn might promote the local immune response.

Polymorphonuclear neutrophils (PMNs) produce an abundance of bactericidal and cytotoxic molecules consistent with their role as first-line defense against bacterial infection. PMNs, however, also cause efficient cellular cytotoxicity when targeted through Fc receptors to appropriate antibody-coated target cells. Although this so-called antibody-dependent cellular cytotoxicity (ADCC) was described many years ago, the mechanism of killing is still elusive. We now have found that PMNs contain perforin and granzyme B, the 2 molecules known as the cytotoxic entity of natural killer cells and of cytotoxic T lymphocytes as well. Lysates of PMNs were lytic for chicken erythrocytes in a time-, temperature-, and Ca2+-dependent manner. Moreover, apoptosis of Jurkat cells was induced, consistent with the observation that the PMN lysates contain enzymatically active granzyme B. Taken together, our data provide evidence for the presence of perforin and granzyme B within the cytotoxic arsenal of PMNs. (Blood. 2004;103:1099-1104)

Shortening of telomeres: Evidence for replicative senescence of T cells derived from patients with Wegener's granulomatosis. Background Replicative senescence describes the fact that somatic cells undergo a finite and predictable number of cell divisions before entering an irreversible state of growth arrest. Progressive shortening of the telomeres, a consequence of cell division, is a reliable indicator of replicative senescence. Method We analyzed telomere length of DNA derived from T cells of patients suffering from Wegener's granulomatosis by Sourthern blotting. Moreover, expression of CD28, another marker for replicative senescence, was tested by cytofluorometry. Results In patients with disease for more than 5 years, short telomeres were detected in addition to telomeres of normal length, indicating replicative senescence of discrete T-cell clones. Reduced expression of CD28 was noted, particularly on CD8-positive T cells, derived from patients with disease for more than 5 years and short telomeres. Conclusion Our data provide evidence that a portion of T cells had undergone replicative senescence, which in turn indicates clonal expansion of T cells as consequence of activation.

The complement receptor 3 (CR3; CD11b/CD18) is present exclusively on leukocytes, particularly on NK cells, monocytes and polymorphonuclear neutrophils. Approximately 10% of peripheral T lymphocytes and, as we found now mainly CD8(+) cells, expressed CD11b. Upon stimulation, however, expression of CD11b was up-regulated also on CD4(+) cells. Stimulation of T cells either by cross-linked anti-CD3 and IL-2 or by mononuclear cells and mitogen yielded up to 28% CD11b(+) T cells. The majority of CD11b(+) T cells also expressed CD56. T cell lines established from healthy donors were also found to express CR3. When restimulated up to 90% of cells became positive for CD11b making those cells an ideal tool for studying the functional role of CD11b. Antibodies to CD11b and bona fide ligands for the complement receptor inhibited the anti-CD3-induced T cell proliferation and as well as IL-2 release. In contrast, proliferation of a CD11b(-) T cell line was not inhibited. Taken together, our data indicate an activation-dependent expression of the complement receptor on T cells and suggest a regulatory function.

Fibronectin synthesis by human tubular epithelial cells in culture: Effects of PDGF and TGF-β on synthesis and splicing. Background Enhanced synthesis of extracellular matrix proteins including fibronectin (FN) is associated with the development of sclerosis. In this context we studied FN synthesis by tubular epithelial cells in response to transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF). Methods FN protein synthesis by human tubular epithelial cells in culture (TEC) was measured by biosynthetic labeling and ELISA. Splicing of FN was assessed by RT-PCR and by Northern blotting. Results Cultivated TEC synthesized and released FN, the majority of which was deposited as an unsoluble protein and a minor portion (10 to 15%) was released into the supernatant. TGF-β and, to a lesser degree, PDGF, up-regulated FN synthesis. All three FN splice variants (EDA, EDB, and IIICS) were produced. PDGF did not influence the splicing. TGF-β preferentially up-regulated the EDA splice variant, but had no effect on the splicing of the other domains. Conclusions PDGF and TGF-β both up-regulate FN synthesis of TEC. TGF-β, but not PDGF, also changed the quality of the de novo synthesized FN, and thus has a different role in the development of sclerosis.

Recent studies show that regulatory T cells (Tregs) play an essential role in tolerance induction after organ transplantation. In order to examine whether there are differences in the composition of the total CD4(+)CD127(low+/-)FoxP3(+)- Treg cell pool between stable transplant patients and patients with biopsy proven rejection (BPR), we compared the percentages and the functional activity of the different Treg cell subsets (DR(high+)CD45RA(-)-Tregs, DR(low+)CD45RA(-)-Tregs, DR(-)CD45RA(-)-Tregs, DR(-)CD45RA(+)-Tregs). All parameters were determined during the three different periods of time after transplantation (0-30 days, 31-1,000 days, >1,000 days). Among 156 transplant patients, 37 patients suffered from BPR. The most prominent differences between rejecting and non-rejecting patients were observed regarding the DR(high+)CD45RA(-)-Treg cell subset. Our data demonstrate that the suppressive activity of the total Treg pool strongly depends on the presence of these Treg cells. Their percentage within the total Treg pool strongly decreased after transplantation and remained relatively low during the first year after transplantation in all patients. Subsequently, the proportion of this Treg subset increased again in patients who accepted the transplant and reached a value of healthy non-transplanted subjects. By contrast, in patients with acute kidney rejection, the DR(high+)CD45RA(-)-Treg subset disappeared excessively, causing a reduction in the suppressive activity of the total Treg pool. Therefore, both the monitoring of its percentage within the total Treg pool and the monitoring of the HLA-DR MFI of the DR(+)CD45RA(-)-Treg subset may be useful tools for the prediction of graft rejection.

Objectives. MMF is cleaved in the acidic milieu of the gastric compartment. However, its absorption might be impeded by proton pump inhibitors (PPIs), which suppress acid production and thus increase stomach pH. Since PPIs are widely used, it is useful to clarify whether the total drug amount of MMF is available in patients undergoing PPI treatment. Methods. We analysed 36 patients with autoimmune diseases under stable MMF maintenance therapy. Twenty-three patients received co-medication with pantoprazole; 13 patients received no treatment with PPIs or antacids. To assess the immunosuppressive potency, we measured mycophenolic acid levels and inosin monophosphate dehydrogenase (IMPDH) activity with a validated HPLC method in plasma samples collected pre-dose and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10 and 12 h after oral administration. Results. The mean MMF dosage of the non-PPI patients was 770 (249) mg/12 h and 771 (291) mg/12 h in pantoprazole-treated patients (NS). The total area under the curve of MMF showed a 37% reduction in PPI patients vs those treated with no PPIs (P < 0.01), and the maximum peak concentration of MMF was 60% lower in the pantoprazole patients (P < 0.001). The MMF exposure correlated with the inhibition of IMPDH activity. The area of enzyme activity curve was 42% higher in the PPI patients (P < 0.01). Conclusions. The co-medication of pantoprazole with MMF significantly influences the drug exposure and immunosuppressive potency of MMF in patients with autoimmune diseases. This finding might at least partly explain the different outcomes in studies using MMF for maintenance therapy.

Regulatory T cells (Tregs) are known to suppress alloimmune responses during pregnancy and post organ transplantation. We demonstrate that a distinct subset of FoxP3(+)DR(+)-Tregs among the total CD4(+)CD127(low+/-)CD25(+)-Treg cell pool is critically involved in preterm labor induction and kidney transplant rejection as well. Compared to healthy pregnancies and non-rejecting kidney recipients, we found that the percentage of the FoxP3(+)DR(+)-Treg subset was not reduced, but that the level of HLA-DR expression of such Tregs was strongly diminished in preterm laboring women and in patients with acute renal allograft rejection. In addition, both patient collectives showed a significantly reduced suppressive activity of their circulating CD4(+)CD127(low+/-)CD25(+)-Treg cell pool. Our findings propose that the FoxP3(+)DR(+)-Treg subset may be decisively responsible for the suppressive activity of the total CD4(+)CD127(low+/-)CD25(+)-Treg cell pool and that the immunologic mechanisms leading to preterm labor necessitating preterm delivery may be similar to those leading to allograft rejection after transplantation.

Renal injury is accompanied by the presence of infiltrating inflammatory cells in the glomerulus and tubulointerstitium. FTY720 modifies lymphocyte migration into injured tissues by lymphocyte sequestration to secondary lymphoid organs. The purpose of this study was to examine the potential of FTY720 to influence the inflammatory response in a nonimmunological model of renal failure. Sham-operated and 5/6 nephrectomized (SNX) Sprague-Dawley rats received two different doses of FTY720 or vehicle orally for 14 wk. Treatment with FTY720 reduced glomerular and tubulointerstitial damage in SNX rats but failed to stabilize creatinine clearance. The increase in gene expression of chemokine receptors CCR1, CCR2, and CCR5 in kidneys of vehicle-treated SNX rats was significantly attenuated by high-dose FTY720. Treatment with high-dose FTY720 tended to normalize RANTES and MCP-1 renal gene expression. FTY720 affected not only glomerular and tubulointerstitial lymphocytes, but M1 and M2 phenotype macrophages were also reduced. FTY720 significantly reduced key mediators of renal inflammation and fibrosis. FTY720 also decreased immunoregulation of M2 macrophages, which are beneficial for tissue remodeling and repair.

Acyl homoserine lactones are synthesized by Pseudomonas aeruginosa as signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that N -(3-oxododecanoyl)- l -homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.

The P. aeruginosa quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) interacts not only with bacteria, but also with mammalian cells, among others with those of the immune defence system. We focussed on the possible interaction of 3OC12-HSL with human polymorphonuclear neutrophils (PMN), because these cells are the first to enter an infected site. We found that 3OC12-HSL attracts PMN, and up-regulates expression of receptors known to be involved in host defence, including the adhesion proteins CD11b/CD18 and the immunoglobulin receptors CD16 and CD64. Furthermore, the uptake of bacteria (phagocytosis), which is crucial for an efficient defence against infection, was enhanced. Thus, recognising and responding to 3OC12-HSL not only attracts the PMN to the site of a developing biofilm, but also reinforces their defence mechanisms, and hence could be a means to control the infection in an early stage and to prevent biofilm formation.

Fibronectin (FN) is a multifunctional extracellular matrix glycoprotein, which participates in cell migration and signalling to adhering cells. Due to alternative splicing and post-translational modifications, different isoforms of FN are generated by a wide variety of cells. T lymphocytes synthesize a surface-associated isoform of FN, containing the two 'extradomains' EDA and EDB. In the present study, we identified gangliosides within the T-cell membrane as specific binding sites for the N-terminal 30 kDa fragment of FN. When T cells were activated with anti-CD3 coated beads, FN, together with the ganglioside GM1, converged at the contact zone. Moreover, endogenous FN was present in the detergent insoluble microdomain. The function of FN in T cells is still under investigation; however, its presence together with gangliosides at the activation site suggests participation in T-cell signalling.

The modulation the specific, adaptive immune response by complement, particularly of by complement C3, is mainly attributed to its interaction with complement receptors on B-lymphocytes. The function of complement receptors on T-lymphocytes, in contrast, is less well understood, although expression of the complement receptor (CR)1 and CR3 on T-cells has been described years ago. In the present study we investigated the effect of antibodies to CR1 on T-cell lines and peripheral T-cells of healthy donors, respectively. Antibodies to CR1 profoundly inhibited the proliferation of the T-cells; of note is, that exogenously added interleukin 2, though enhancing proliferation, did not overcome the inhibitory effect mediated by anti-CR1. While anti-CR1 had no effect on the activation of the immediate early genes c-jun or c-fos nor on the early increase of gamma interferon- or interleukin 2-specific RNA, the protein synthesis of those cytokines was inhibited. Moreover, synthesis of the proliferating cell nuclear antigen (PCNA) was reduced as was the expression of cyclins, particularly of cyclin A and cyclin D3. Taken together, the data indicate that triggering CR1 inhibits proliferation of T-lymphocytes by a mechanism operating downstream of the initial signalling events.

In polymorphonuclear neutrophils (PMN) CD14, one of the receptors for lipopolysaccharides (LPS) is stored intracellularly as a preformed protein, with only few receptors expressed on the surface. We now report that in patients with severe bacterial infections, CD14 expression is profoundly upregulated, as is CD64 (FcgammaRI), the high-affinity receptor for IgG, whereas CD16 (FcgammaRIII) was partly lost from the surface. To further analyze regulation of these receptors, PMN of healthy donors were exposed to low doses of LPS. By brief exposure (10-120 min) to LPS, CD14 was transferred to the surface in a cytochalasin B-sensitive manner, as were CD16 and CD64. Prolonged culture (up to 48 h) resulted in a further upregulation of CD14, sustained expression of CD64, and profound decline of CD16, yielding a similar pattern of receptor expression as seen in the patients. Subsequent studies revealed that LPS induced de novo synthesis of CD14: the increase of surface expression could be inhibited by cycloheximide and by interfering with a known LPS-induced signaling event, the translocation of NFkappaB. Moreover, an up to 10-fold increase of specific mRNA was seen, as was incorporation into CD14 of 35S-methionine. The de novo synthesis prolonged expression of CD14, whereas the CD16 expression declined, generating a PMN phenotype characteristic for severe infection and indicative of escape from apoptosis of a PMN subpopulation.

Summary Upon cultivation with interferon-γ (IFN-γ) and granulocyte/macrophage-colony stimulating factor (GM-CSF) polymorphonuclear neutrophils (PMN) acquire characteristics of dendritic cells, including expression of major histocompatibility complex (MHC) class II antigens, of the co-stimulatory antigens CD80, CD86 and of CD83, the latter considered to be specific for dendritic cells. Dendritic-like PMN were also able to present to T cells antigens in a MHC class II-restricted manner. To assess whether dendritic-like PMN are also generated in vivo, cells of patients with acute bacterial infections and of patients with chronic inflammatory diseases (primary vasculitis) were tested. During acute infection up to 80% of PMN acquired CD83, but remained negative for MHC class II, CD80 or CD86. PMN of patients with primary vasculitis expressed MHC class II antigens, CD80 and CD86, but not CD83, indicating that up-regulation of MHC class II and of CD83 are not necessarily linked to each other. Indeed, parallel studies with PMN of healthy donors showed that while IFN-γ and granulocyte/macrophage colony stimulating factor (GM-CSF) induced both, MHC class II and CD83, tumour necrosis factor (TNF)-α selectively induced de novo synthesis of CD83. The function of CD83 on PMN is still elusive. A participation in the MHC class II-restricted antigen presentation could be ruled out, consistent with the segregation of MHC class II and CD83 expression. Regardless, however, of its function, CD83 expression could serve as a marker to differentiate between acute and chronic inflammation.

The terminal complement complex C5b-9 is known to participate in inflammatory processes including glomerular or tubulointerstitial injury. Injury appears to be a direct consequence of C5b-9-mediated cell stimulation. In that context we studied activation of tubular epithelial cells by C5b-9 particularly with regard to fibronectin synthesis and the transmembrane signals involved. C5b-9 in sublytic concentrations caused a rise of intracellular calcium and of cAMP, followed by an increase in abundance of fibronectin-specific mRNA and accumulation of protein. Stabilized cAMP or increasing the cAMP level by forskolin enhanced fibronectin synthesis with similar kinetics. The effect of cAMP could be enhanced by adding a calcium ionophore. Since the fibronectin gene is known to have a cAMP-responsive element, the data suggest that C5b-9 increases fibronectin synthesis via generation of cAMP.

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in beta 2-micro-globulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharide L-fucose suggesting that dialysis membrane associated L-fucose residues are involved in leukocyte activation. In this study we have detected and quantitated L-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detected L-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3 +/- 3.6 to 90.2 +/- 5.0 pmol cm-2 for Hemophan or Cuprophan respectively. Enzymatic cleavage of terminal alpha-L-fucopyranoses with alpha-L-fucosidase yielded 7.7 +/- 3.3 pmol L-fucose per cm2 for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts of L-fucose. In a second approach, binding of the fucose specific lectins of Lotus tetragonolobus and Ulex europaeus (UEAI) demonstrated the presence of biologically accessible L-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan, and up to 2.6 +/- 0.3 pmol L-fucose per cm2 was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associated L-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.

Contact of mononuclear cells (MNC) with cuprophan membranes in vitro causes an increase in beta 2-microglobulin (beta 2m) synthesis. Since in vivo the dialyzer membrane is rapidly coated with plasma proteins, contact activation of MNC was tested in the presence of normal human serum (NHS). After contact with cuprophan, deposition of C5b-9 on the cells was seen, followed by an increase in beta 2m synthesis and cytokine release, exceeding that seen after contact activation in the absence of serum. Inactivated serum or serum deficient in C8 did not increase beta 2m production, indicating that the additional activation was due to complement C5b-9. The results suggest that there are two cuprophan-related mechanisms of cell activation: one by contact of cells with the membrane, the other by the complement activation products. Both might synergistically contribute to an increased beta 2m synthesis in hemodialysis patients.