Azithromycin and the treatment of lymphocytic airway inflammation after lung transplantation.

Lymphocytic airway inflammation is a major risk factor for chronic lung allograft dysfunction, for which there is no established treatment. We investigated whether azithromycin could control lymphocytic airway inflammation and improve allograft function. Fifteen lung transplant recipients demonstrating acute allograft dysfunction due to isolated lymphocytic airway inflammation were prospectively treated with azithromycin for at least 6 months (NCT01109160). Spirometry (FVC, FEV1 , FEF25-75 , Tiffeneau index) and FeNO were assessed before and up to 12 months after initiation of azithromycin. Radiologic features, local inflammation assessed on airway biopsy (rejection score, IL-17(+) cells/mm(2) lamina propria) and broncho-alveolar lavage fluid (total and differential cell counts, chemokine and cytokine levels); as well as systemic C-reactive protein levels were compared between baseline and after 3 months of treatment. Airflow improved and FeNO decreased to baseline levels after 1 month of azithromycin and were sustained thereafter. After 3 months of treatment, radiologic abnormalities, submucosal cellular inflammation, lavage protein levels of IL-1ß, IL-8/CXCL-8, IP-10/CXCL-10, RANTES/CCL5, MIP1-α/CCL3, MIP-1ß/CCL4, Eotaxin, PDGF-BB, total cell count, neutrophils and eosinophils, as well as plasma C-reactive protein levels all significantly decreased compared to baseline (p < 0.05). Administration of azithromycin was associated with suppression of posttransplant lymphocytic airway inflammation and clinical improvement in lung allograft function.

HylA, an alternative hydrolase for initiation of catabolism of the phenylurea herbicide linuron in Variovorax sp. strains.

Variovorax sp. strain WDL1, which mineralizes the phenylurea herbicide linuron, expresses a novel linuron-hydrolyzing enzyme, HylA, that converts linuron to 3,4-dichloroaniline (DCA). The enzyme is distinct from the linuron hydrolase LibA enzyme recently identified in other linuron-mineralizing Variovorax strains and from phenylurea-hydrolyzing enzymes (PuhA, PuhB) found in Gram-positive bacteria. The dimeric enzyme belongs to a separate family of hydrolases and differs in Km, temperature optimum, and phenylurea herbicide substrate range. Within the metal-dependent amidohydrolase superfamily, HylA and PuhA/PuhB belong to two distinct protein families, while LibA is a member of the unrelated amidase signature family. The hylA gene was identified in a draft genome sequence of strain WDL1. The involvement of hylA in linuron degradation by strain WDL1 is inferred from its absence in spontaneous WDL1 mutants defective in linuron hydrolysis and its presence in linuron-degrading Variovorax strains that lack libA. In strain WDL1, the hylA gene is combined with catabolic gene modules encoding the downstream pathways for DCA degradation, which are very similar to those present in Variovorax sp. SRS16, which contains libA. Our results show that the expansion of a DCA catabolic pathway toward linuron degradation in Variovorax can involve different but isofunctional linuron hydrolysis genes encoding proteins that belong to evolutionary unrelated hydrolase families. This may be explained by divergent evolution and the independent acquisition of the corresponding genetic modules.

Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity.

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family.

Cytokine-stimulated human osteosarcoma cells (MG-63) secrete several related chemotactic factors, including the neutrophil-activating protein interleukin 8 (IL-8) and the monocyte chemotactic protein (MCP)-1. We describe the isolation and characterization of two novel monocyte chemotactic factors from this tumor cell line. Although these proteins copurified with MCP-1 and IL-8 on heparin-Sepharose, they could be separated by cation-exchange fast protein liquid chromatography and reverse-phase high-performance liquid chromatography. The corresponding 7.5- and 11-kD proteins were NH2-terminally blocked but were identified by sequencing peptide fragments. They showed a primary structure mostly related to that of MCP-1 and were therefore designated MCP-2 and MCP-3, respectively. These molecules can be classified in a subfamily of proinflammatory proteins characterized by the conservation of cysteine residues. MCP-2 and MCP-3 are also functionally related to MCP-1 because they specifically attract monocytes, but not neutrophils, in vitro. The chemotactic potency (specific activity) was comparable for all three MCPs. Intradermal injection of these proteins in rabbits resulted in selective monocyte recruitment in vivo. Since tumor cells are good producers of leukocyte chemotactic factors, it could be questioned whether these molecules can indirectly control tumor growth by attracting leukocytes or whether they rather promote invasion by the secretion of proteases from the attracted cells.

Dysbiotic Biofilms Deregulate the Periodontal Inflammatory Response.

Periodontal diseases originate from a dysbiosis within the oral microbiota, which is associated with a deregulation of the host immune response. Although little is known about the initiation of dysbiosis, it has been shown that H2O2 production is one of the main mechanisms by which some commensal bacteria suppress the outgrowth of pathobionts. Current models emphasize the critical nature of complex microbial biofilms that form unique microbial ecologies and of their change during transition from health (homeostatic) to disease (dysbiotic). However, very little is known on how this alters their virulence and host responses. The objective of this study was to determine differences in virulence gene expression by pathobionts and the inflammatory host response in homeostatic and dysbiotic biofilms originating from the same ecology. Quantitative polymerase chain reaction was performed to quantify the pathobiont outgrowth. Expression analysis of bacterial virulence and cellular inflammatory genes together with cytokine enzyme-linked immunosorbent assays were used to detect differences in bacterial virulence and to analyze potential differences in inflammatory response. An increase in pathobionts in induced dysbiotic biofilms was observed compared to homeostatic biofilms. The main virulence genes of all pathobionts were upregulated in dysbiotic biofilms. Exposure of these dysbiotic biofilms to epithelial and fibroblast cultures increased the expression of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, and matrix metalloprotease 8, but especially the chemokine CXCL8 (IL-8). Conversely, homeostatic and beneficial biofilms had a minor immune response at the messenger RNA and protein level. Overall, induced dysbiotic biofilms enriched in pathobionts and virulence factors significantly increased the inflammatory response compared to homeostatic and commensal biofilms.

Monocyte chemotactic protein-1 (MCP-1), -2, and -3 are chemotactic for human T lymphocytes.

Monocyte chemotactic protein (MCP)-1, -2, and -3 all have been shown to induce monocyte/macrophage migration in vitro and MCP-1, also known as MCAF, chemoattracts basophils and mast cells. We report here that natural MCP-1 as well as synthetic preparations of MCP-2 and MCP-3 stimulate significant in vitro chemotaxis of human peripheral blood T lymphocytes. This MCP-induced migration was dose-dependent and directional, but not chemokinetic. Phenotypic analysis of the T cell population responsive to MCP-1, MCP-2, and MCP-3 demonstrates that both CD4+ and CD8+ T cells migrated in response to these chemokines. Similar results were observed using human CD4+ and CD8+ T cell clones. Neutralizing antisera to MCAF or MCP-2 abrogated T cell migration in response to MCP-1 and MCP-2, respectively, but not to RANTES. Subcutaneous administration of purified MCP-1 into the hind flanks of SCID mice engrafted with human peripheral blood lymphocytes (PBL) induced significant human CD3+ T cell infiltration into the site of injection at 4 h. These results demonstrate that MCP-1, MCP-2, and MCP-3 are inflammatory mediators that specifically stimulate the directional migration of T cells as well as monocytes and may play an important role in immune cell recruitment into sites of antigenic challenge.

The LD78beta isoform of MIP-1alpha is the most potent CCR5 agonist and HIV-1-inhibiting chemokine.

LD78alpha and LD78beta are 2 highly related nonallelic genes that code for different isoforms of the human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). Two molecular forms of natural LD78beta (7.778 and 7.793 kDa) were identified from conditioned media of stimulated peripheral blood mononuclear cells. Although LD78alpha and LD78beta only differ in 3 amino acids, both LD78beta variants were 100-fold more potent chemoattractants for mouse lymphocytes than was LD78alpha. On the contrary, LD78beta was only 2-fold more efficient than LD78alpha in chemoattracting human lymphocytes and monocytes. Using CC chemokine receptor-transfected cells, both molecular forms of LD78beta proved to be much more potent than LD78alpha in inducing an intracellular calcium rise through CCR5. Compared with LD78alpha and RANTES, this preferential binding of LD78beta to CCR5 resulted in a 10- to 50-fold higher potency in inhibiting infection of peripheral blood mononuclear cells by CCR5-using (R5) HIV-1 strains. To date, LD78beta is the most potent chemokine for inhibiting HIV-1 infection, and can be considered as a potentially important drug candidate for the treatment of infection with R5 HIV-1 strains.

Oligosaccharides of recombinant mouse gelatinase B variants.

Gelatinase B (matrix metalloproteinase-9, MMP-9) contains three N-glycosylation sites and a Ser/Thr/Pro-rich type V collagen domain with repetitive attachment sites for O-linked sugars. Recombinant mouse gelatinase B was expressed in the yeast Pichia pastoris and the N-linked oligosaccharides of the truncated glycoprotein variants were analysed by in gel enzymatic release followed by mass spectrometry and normal phase HPLC. This technology, despite of the limiting amount of material, allowed the analysis of the formula of N- and O-linked sugars of the different glycoprotein variants. The 112/99- and 88-kDa gelatinase B forms each contained an oligomannose series (Man8GlcNAc2 to Man15GlcNAc2). Analysis of the hydrazine-released sugars showed that the O-linked oligosaccharides contained alpha1-2, alpha1-3 or alpha1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants.

Purification and Identification of Natural Chemokines

A novel family of chemotactic cytokines or chemokines, essential for the directed migration of leukocytes to sites of inflammation, has been identified during the past decade. To obtain microgram amounts of natural chemokines, normal (e.g., freshly isolated leukocytes, connective tissue cell cultures) or malignant cell lines have to be selectively induced with endogenous (cytokines) or exogenous (bacterial, viral, or plant) products. We have developed a four-step procedure that allows for the complete purification of active C-C (MCP-1, MCP-2, MCP-3, RANTES, MIP-1alpha and MIP-1beta) and C-X-C (IL-8, GRO-alpha, GRO-beta, GRO-gamma, GCP-2, ENA-78, IP-10, PF-4, and CTAPIII/betaTG/NAP-2) chemokines from bulk volumes of culture supernatant. This method is applicable for the isolation of recombinant chemokines. Conditioned medium was first concentrated and partially purified on silicic acid or controlled pore glass beads. Further purification to homogeneity was achieved using heparin-Sepharose or antibody affinity chromatography, cation exchange FPLC, and reverse-phase HPLC. Purification of chemokines was monitored by testing column fractions for biological (chemotaxis) or immuno (RIA, ELISA) activity and protein content (SDS-PAGE). Homogeneous proteins were identified by amino-terminal or internal protein sequence analysis.

Human monocyte chemotactic proteins-2 and -3: structural and functional comparison with MCP-1.

Structurally, the monocyte chemotactic proteins MCP-1, -2, and -3 form a subfamily of the C-C or beta-chemokines. Like other chemokines, MCPs are produced by a variety of cells on stimulation with cytokines (interleukin-1, tumor necrosis factor-alpha, interferon-gamma), bacterial and viral products or mitogens. MCP-1 levels are enhanced during infection and inflammation, which are characterized by leukocyte infiltration. In vitro, MCPs are chemotactic for a distinct spectrum of target cells and show different specific biological activities depending on the cell type and the chemokine tested. MCP-3 has the broadest range in that it activates monocytes, dendritic cells, lymphocytes, natural killer cells, eosinophils, basophils, and neutrophils. The most sensitive cells to all three MCPs are lymphocytes and monocytes. MCP-1 is a potent basophil activator but does not attract eosinophils, whereas, at higher concentrations, MCP-2 also stimulates both eosinophils and basophils. The signal transduction of MCPs on monocytes involves at least two G protein-linked C-C chemokine receptors: C-C CKR-1 binds MCP-3 and C-C CKR-2 binds MCP-1 and MCP-3 but not MCP-2. Receptor binding leads to enhanced [Ca2+]i for all chemokines except for MCP-2.

Synergistic induction of MCP-1 and -2 by IL-1beta and interferons in fibroblasts and epithelial cells.

Monocyte chemotactic protein (MCP)-1 and MCP-2, two closely related CC chemokines, are important mediators of monocyte and lymphocyte migration. These chemokines are secreted by various normal cell types, including fibroblasts, epithelial cells, and leukocytes, as well as by tumor cells. After stimulation with different cytokines and cytokine inducers the MCP-2 production levels are always lower than those of MCP-1. In human diploid fibroblasts cytokines differentially regulate chemokine induction, interleukin (IL)-1beta and interferon (IFN)-gamma being potent stimuli of MCP-1 and MCP-2, respectively. Co-stimulation of fibroblasts by 10 U/mL IL-1beta and 20 ng/mL IFN-gamma resulted in a synergistic induction of MCP-2, whereas the combined effect on MCP-1 and IL-6 production was rather additive. These findings were confirmed at the mRNA level by Northern blot analysis. In contrast, in human MG-63 fibroblastoid cells and HEp-2 epithelial cells, selected for their poor responsiveness to IL-1beta and IFN-gamma, MCP-2 as well as MCP-1 and IL-6 were synergistically induced, yielding protein levels that were increased 3- to 30-fold above the additive levels. When IFN-beta was used as a co-stimulant of IL-1beta, a similar synergistic induction of MCP-1 and MCP-2 was measured both at the protein and the mRNA level. It can be concluded that, when synergy occurred, the MCP-1 and MCP-2 expression levels reached a comparable maximum, indicative for an equal contribution of these chemokines in normal and pathological conditions.

Granulocyte chemotactic protein-2 and related CXC chemokines: from gene regulation to receptor usage.

Chemokines contribute to the inflammatory response by selective attraction of various leukocytic cell types. Human GCP-2 was originally identified by amino acid sequence analysis as a CXC chemokine co-produced with IL-8 by osteosarcoma cells. Furthermore, the complete coding domain of human GCP-2 was disclosed by means of RT-PCR. Similarly, mouse GCP-2 was isolated from fibroblastoid and epithelial cells and completely identified by sequence analysis. Human and mouse GCP-2 share 61% identical amino acids. Both chemokines occur as multiple NH2-terminally truncated forms. The shorter forms of mouse, but not those of human, GCP-2 showed a higher neutrophil chemotactic potency and gelatinase B releasing capacity. Mouse GCP-2 was a more potent neutrophil activator than human GCP-2, natural mouse KC, and MIP-2. Human GCP-2 was not chemotactic for monocytes, lymphocytes, or eosinophils. Quantitative studies of mRNA expression in diploid fibroblasts revealed GCP-2 induction by IL-1beta. Human GCP-2 induced [Ca2+]i increase in neutrophils, which was reciprocally desensitized by IL-8, GROalpha, and ENA-78. Human GCP-2 induced [Ca2+]i increases and chemotactic responses in both CXCR1- and CXCR2-transfected cells. Finally, GCP-2 provoked neutrophil accumulation and plasma extravasation in rabbit skin. In humans, GCP-2 complements the activity of IL-8 as neutrophil chemoattractant and activator but it constitutes a major neutrophil chemokine in the mouse. GCP-2 induces neutrophil chemotaxis and activation but it might also contribute to detrimental tissue damage in sepsis, acute respiratory distress syndrome, acute hypersensitivity reactions, and autoimmune diseases. It might also influence the invasive capacity of GCP-2-secreting tumor cells.

Gelatinase B functions as regulator and effector in leukocyte biology.

Matrix metalloproteinases (MMPs) form a family of enzymes with major actions in the remodeling of extracellular matrix (ECM) components. Gelatinase B (MMP-9) is the most complex family member in terms of domain structure and regulation of its activity. Gelatinase B activity is under strict control at various levels: transcription of the gene by cytokines and cellular interactions; activation of the pro-enzyme by a cascade of enzymes comprising serine proteases and other MMPs; and regulation by specific tissue inhibitors of MMPs (TIMPs) or by unspecific inhibitors, such as alpha2-macroglobulin. Thus, remodeling ECM is the result of the local protease load, i.e., the net balance between enzymes and inhibitors. Glycosylation has a limited effect on the net activity of gelatinase B, and in contrast to the all-or-none effect of enzyme activation or inhibition, it results in a higher-level, fine-tuning effect on the ECM catalysis by proteases in mammalian species. Fast degranulation of considerable amounts of intracellularly stored gelatinase B from neutrophils, induced by various types of chemotactic factors, is another level of control of activity. Neutrophils are first-line defense leukocytes and do not produce gelatinase A or TIMP. Thus, neutrophils contrast sharply with mononuclear leukocytes, which produce gelatinase A constitutively, synthesize gelatinase B de novo after adequate triggering, and overproduce TIMP-1. Gelatinase B is also endowed with functions other than cleaving the ECM. It has been shown to generate autoimmune neo-epitopes and to activate pro-IL-1beta into active IL-1beta. Gelatinase B ablation in the mouse leads to altered bone remodeling and subfertility, results in resistance to several induced inflammatory or autoimmune pathologies, and indicates that the enzyme plays a crucial role in development and angiogenesis. The major human neutrophil chemoattractant, IL-8, stimulates fast degranulation of gelatinase B from neutrophils. Gelatinase B is also found to function as a regulator of neutrophil biology and to truncate IL-8 at the amino terminus into a tenfold more potent chemokine, resulting in an important positive feedback loop for neutrophil activation and chemotaxis. The CXC chemokines GRO-alpha, CTAP-III, and PF-4 are degraded by gelatinase B, whereas the CC chemokines MCP-2 and RANTES are not cleaved.

The soluble extracellular portion of the human interferon-gamma receptor is a valid substitute for evaluating binding characteristics and for neutralizing the biological activity of this cytokine.

In order to have a reliable and reproducible source of soluble human interferon-gamma (HuIFN-gamma) receptor at our disposal both for studying binding phenomena and for evaluating its neutralizing potential towards the cytokine, we expressed the extracellular part of the receptor in J558L mouse myeloma cells as a fusion protein with the C-terminal c-myc TAG (HuECR-gamma-TAG). It is expected that the receptor will undergo post-translational modifications comparable to that in humans. The affinity purified soluble receptor was subjected to mass spectrometry analysis resulting in a molecular size of 31 to 40 kDa and showed heterogeneous N-glycosylation with an M(r)-contribution of 4 to 13 kDa. Its HuIFN-gamma binding affinity, determined by real time biospecific interaction (BIAcore) analysis, resulted in a value of Kd = 2 x 10(-9) M, which is in agreement with the high affinity described for the cell anchored complete HuIFN-gamma receptor (Kd = 5-35 x 10(-9) M). HuECR-gamma-TAG was able to neutralize the biological activity of HuIFN-gamma in an in vitro antiviral assay. Furthermore, we report for the first time the association and dissociation rate constants, which were, respectively, 2.4 x 10(5) M-1 s-1 and 4.8 x 10(-4) s-1. In conclusion, this mammalian source of the extracellular soluble HuIFN-gamma receptor represents a valuable tool for extensive in vitro studies of the HuIFN-gamma receptor interaction. Furthermore, in view of its expected low or nonimmunogenicity it opens new ways for immunomodulation in vivo.

In vitro evidence that an 11-kilodalton N-terminal fragment of proopiomelanocortin is a growth factor specifically stimulating the development of lactotrophs in rat pituitary during postnatal life.

We have previously shown that in reaggregate cell cultures of 14-day-old female rat pituitary, LHRH is capable of stimulating lactotrophs to enter the S-phase of the cell cycle, and that this effect is mediated by paracrine growth factors secreted from gonadotrophs. In the present report we describe the isolation, purification, and chemical characterization of one of these growth factors. Gonadotroph-rich aggregates from 14-day-old female rats were cultured for 6-7 weeks in serum-free and serum albumin-free defined medium in the presence of 0.01 nM LHRH. A total volume of 3.2 liters containing material secreted from 2.5 x 10(8) cells was batchwise concentrated on Sep-Pak C18/125 A cartridges (10 g) and retained molecules ultrafiltrated on Centricon 3 membrane filters [mol wt (M(r)) cut-off, 3 kilodaltons (kDa)]. Material capable of increasing the number of [3H]thymidine ([3H]T)-incorporating lactotrophs in pituitary cell aggregates of 2-week-old rats was isolated by chromatography on two reverse phase HPLC columns, one HPLC gel filtration column, and a final reverse phase HPLC column. A substance was obtained which, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, ran as a single band with an apparent M(r) of 16 kDa. On gel filtration, the apparent M(r) was 11 kDa. N-Terminal amino acid sequence analysis revealed that the substance was a peptide; a sequence of 10 residues was obtained, which was identical to that in the N-terminal part of rat POMC. By electrospray ionization mass spectrometry, six different compounds with slightly different M(r), ranging from 10,796-11,108 daltons, were detected. The latter data suggest that the peptide extends C-terminally at least to residue 74, which in the POMC sequence is flanked by an Arg-Arg dibasic residue, a posttranslational cleavage site. The substance increased the number of [3H]T-incorporating lactotrophs in pituitary cell aggregates without affecting [3H]T labeling of other pituitary cell types. Authentic human POMC-(1-76), at a concentration of 10 nM provoked a similar stimulation of the [3H]T-labeled lactotroph number without affecting other cell types. It is concluded that POMC-(1-74) is a growth factor that specifically targets lactotrophs during postnatal development of the rat anterior pituitary.

Efficient secretion of biologically active mouse tumor necrosis factor alpha by Streptomyces lividans.

We have studied the production of mouse tumor necrosis factor alpha (mTNF) with Streptomyces lividans as host. mTNF cDNA was fused to the alpha-amylase-encoding gene (aml) of Streptomyces venezuelae ATCC15068 at 12 amino acids (aa) downstream from the signal-peptidase cleavage site so that the aa surrounding this processing site were conserved. S. lividans containing this construct secreted mTNF at moderately high levels (1-10 micrograms/ml) as a biologically active compound of high specific activity (1 x 10(8) units/mg protein). No unprocessed pre-protein and virtually no processed protein could be detected in the cell lysates. N-terminal aa sequence analysis indicated microheterogeneity (-3 to -6 forms) at the N-terminal site of secreted mTNF. It was demonstrated that this microheterogeneity was due to aminopeptidase activity.

Natural human monocyte gelatinase and its inhibitor.

Gelatinases produced by stimulated peripheral blood monocytes were detected by substrate zymography and were compared with those derived from tumor cells. Stimulated monocytes were found to produce an 85 kDa gelatinase which co-migrated upon electrophoretic separation and cross-reacted in immunoprecipitation experiments with a phorbol ester inducible metalloprotease from human tumor cells. The intact natural gelatinase (85 kDa), a high molecular weight and complexed gelatinase as well as a proteolytic fragment (25 kDa) were purified by substrate- and antibody-affinity chromatography techniques. Aminoterminal sequence analysis showed that natural monocyte gelatinase occurs as a truncated form of tumor cell gelatinase/type IV collagenase. Furthermore, peripheral blood monocytes were found to also produce a tissue inhibitor of metalloproteases (TIMP). TIMP was co-purified with gelatinase on gelatin sepharose and identified by microsequencing. The balanced and regulated production of gelatinase and TIMP might be important in monocyte migration and tissue remodeling.

Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion.

Dipeptidyl-peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady-state kinetics of DPPIV/CD26-mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating peptide (PACAP27 and PACAP38), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C-terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.

Processing by CD26/dipeptidyl-peptidase IV reduces the chemotactic and anti-HIV-1 activity of stromal-cell-derived factor-1alpha.

The chemokine stromal-cell-derived factor-1alpha (SDF-1alpha) chemoattracts lymphocytes and CD34+ haematopoietic progenitors and is the ligand for CXCR4 (CXC chemokine receptor 4), the main co-receptor for T-tropic HIV-1 strains. SDF-1alpha was NH2-terminally cleaved to SDF-1alpha(3-68) by dipeptidyl-peptidase IV (CD26/DPP IV), which is present in blood in soluble and membrane-bound form. SDF-1alpha(3-68) lost both lymphocyte chemotactic and CXCR4-signaling properties. However, SDF-1alpha(3-68) still desensitized the SDF-1alpha(1-68)-induced Ca2+ response. In contrast to CD26/DPP IV-processed RANTES(3-68), SDF-1alpha(3-68) had diminished potency to inhibit HIV-1 infection. Thus, CD26/DPP IV impairs the inflammatory and haematopoietic potency of chemokines but plays a dual role in AIDS.

Expression of a human mutant monocyte chemotactic protein 3 in Pichia pastoris and characterization as an MCP-3 receptor antagonist.

The cDNA encoding human monocyte chemotactic protein 3 (hMCP-3) was cloned in pHIL-S1, a vector designed for inducible secreted heterologous expression in the methylotrophic yeast Pichia pastoris. After transformation of P. pastoris by electroporation, several clones with the human MCP-3 gene integrated at the alcohol oxidase (AOX-1) locus were isolated. One of these clones (M30) expressed the mature MCP-3 protein with three additional amino acids at its NH2 terminus as a secretion product in the supernatant. The recombinant protein comigrated on SDS-PAGE and cross-reacted immunologically with synthetic hMCP-3. Intermediate-scale production in shake flasks was obtained at expression levels of approximately 1 mg per liter. The recombinant mutant MCP-3 was purified to homogeneity by adsorption on silicic acid, affinity chromatography on heparin-Sepharose, and reversed-phase HPLC. At the amino terminus of the purified recombinant protein, the presence of the additional sequence Arg-Glu-Phe was confirmed by direct protein sequence analysis. The recombinant hMCP-3 mutein was not glycosylated, as evidenced by deglycosylation experiments and by mass spectrometry. In analogy with MCP-1, the amino terminus of MCP-3 is crucial for its agonistic effect on receptive cells. At concentrations up to 3.5 micrograms/ml, the recombinant mutein was not active in vitro as a chemotactic factor for monocytes. However, the mutant MCP-3 acted as an MCP-3 receptor antagonist in a competition chemotaxis assay at 100- to 1000-fold excess over the synthetic MCP-3 agonist. It might thus be a useful tool to study antagonism of MCP-3 action in vitro and in disease models of cancer and inflammation.