Immunodetection of the murine chemotactic protein CP-10 in bleomycin-induced pulmonary injury.

The murine S-100 protein designated CP-10 is a potent chemotactic factor for phagocytic cells, exhibiting optimal activity in the picomolar range. We assessed the role of this cytokine in the inflammatory response to pulmonary injury following intratracheal administration of bleomycin to mice. In the lungs of normal animals, strong cytoplasmic immunostaining for CP-10 was demonstrable in all recognisable neutrophil leucocytes sequestered within alveolar capillaries. Following induction of pulmonary inflammation in susceptible C57BL/6 mice, numerous CP-10-positive neutrophils were observed, but many of the recruited neutrophils did not exhibit staining for CP-10. No other cells were immunoreactive. The concentration of CP-10 in bronchoalveolar lavage (BAL) fluids from normal mice and mice administered intratracheal saline was below the level of detection by enzyme immunoassay. In contrast, nanomolar levels of CP-10 were detected in unconcentrated BAL fluids from C57BL/6 mice after bleomycin-induced injury, and the presence of monomeric CP-10 was demonstrable by Western blotting. Elevation of CP-10 levels correlated with the influx of inflammatory cells in C57BL/6 mice, but was not demonstrable in BAL fluids from BALB/c mice, which are resistant to pulmonary injury by bleomycin. We conclude that CP-10 may contribute to the recruitment of inflammatory cells in bleomycin-induced lung damage.

Induction of the chemotactic S100 protein, CP-10, in monocyte/macrophages by lipopolysaccharide.

The murine S100 protein CP-10 is a potent chemotactic factor for murine and human myeloid cells in vivo and in vitro. This is the first report describing regulations of the CP-10 gene by a proinflammatory stimulus, lipopolysaccharide (LPS), in cells of the monocyte/macrophage lineage. Murine monocyte/macrophage-like WEHI 265 and RAW 264.7 cells preexposed to 5 to 50 ng/mL LPS expressed significant levels of CP-10 mRNA 4 hours, and maximal at 20 hours, after a secondary LPS challenge. This was accompanied by increasing levels of cell-associated and released CP-10 protein. In contrast, a single dose of LPS upregulated CP-10 mRNA in elicited peritoneal macrophages, whereas mRNA and protein levels decreased following LPS challenge. The state of macrophage differentiation may control responsiveness as LPS had no effect on CP-10 basal levels in bone marrow derived macrophages. LPS-induced CP-10 expression was controlled at the transcriptional level and nuclear run-on and protein synthesis inhibition assays indicated that LPS priming and challenge of RAW cells occurred via distinct pathways. MRP14, another S100 protein generally coordinately expressed with human MRP8, was not induced by LPS under the same conditions. We propose that CP-10 may play a key role in recruitment of leukocytes into tissues in response to gram-negative bacterial infection.

S100 protein CP-10 stimulates myeloid cell chemotaxis without activation.

Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, "classical chemoattractants" such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and "pure chemoattractants" such as TGF-beta 1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the "non-classical" group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca2+]i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca(2+)-independent G-protein-coupled pathway for post-receptor signal transduction triggered by "pure chemoattractants." The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds.

Identification of a chemotactic domain of the pro-inflammatory S100 protein CP-10.

We previously reported the purification and partial amino acid sequence of a novel murine cytokine designated CP-10, which has chemotactic activity for murine polymorphonuclear cells (PMN) and macrophages. The complete cDNA encoding an 88-amino acid polypeptide has been isolated and the sequence is presented here. Transient transfection of CP-10 cDNA into CV-1 cells confirmed the chemotactic activity of rCP-10 for murine PMN. CP-10 has sequence homology with members of the S100 family of Ca(2+)-binding proteins with pronounced amino acid sequence similarities within the putative N- and C-terminal Ca(2+)-binding sites, but differences within their connecting hinge and C-terminal regions. We have confirmed the hypothesis of Kligman and Hilt that functional specificity of individual members of the S100 protein family may reside in the hinge region. A synthetic peptide corresponding to the hinge region of CP-10 (CP-10(42-55) was compared with native CP-10 in chemotaxis and skin test assays. Native CP-10 had potent activity for phagocytic cells, but not lymphocytes, in vitro (optimal activity, 10(-11) to 10(-13) M) and elicited a sustained recruitment of neutrophils and mononuclear cells over 24 h in vivo. The hinge-region peptide had strong chemotactic activity for murine phagocytic cells (optimal activity, 10(-10) - 10(-11) M) but elicited only a transient infiltration of neutrophils over 4 to 8 h after intradermal injection. Results indicate that although the hinge region contributes significantly to the functional specificity of the S100 protein CP-10, sustained cellular recruitment typical of a delayed type hypersensitivity response is apparently dependent on the structural integrity of the protein.

Purification and structural analysis of a murine chemotactic cytokine (CP-10) with sequence homology to S100 proteins.

In delayed-type hypersensitivity reactions, cytokine-mediated cell migration leads to localized accumulation of neutrophils and mononuclear cells over 4-48 h. In contrast to transient (2-6 h) responses elicited by other chemotactic factors, earlier studies indicated that a chemotactic activity previously described in our laboratory elicited skin test responses over 24 h, identical to those induced by injection of antigen into a sensitized test subject. We have isolated this factor, a 10.3-kDa chemotactic protein designated CP-10, from supernatants of activated murine spleen cells. Purification to homogeneity was achieved using affinity chromatography on iminodiacetic acid-immobilized copper and cation-exchange, mixed mode (cation exchange/metal affinity), reversed-phase, and size-exclusion high performance liquid chromatography. CP-10 had maximal chemotactic activity for neutrophils at 10(-13) M. The 76-amino acid sequence, obtained by automated N-terminal microsequence analysis of native CP-10, and fragments derived from trypsin digestion and cyanogen bromide cleavage indicated no sequence identity with any known cytokine or chemotactic factor but revealed up to 55% sequence homology with S100, Ca2(+)-binding proteins. CP-10 appears to be the first protein of this family with a well defined function affecting cell migration, and its biological potency suggests an important role for this cytokine in cellular immune reactions.

Clinical experiences with human parathyroid hormone 1-34.

Human parathyroid hormone (PTH) 1-34 was given to nine normal subjects and to 10 patients with hypoparathyroidism. There were no side effects associated with the protocol employed. In normal subjects, five statistically significant changes occurred during the period of observation: plasma cyclic adenosine monophosphate (cAMP) rose by a factor of 3 (at 30 min), nephrogenous cyclic AMP rose approximately 40-fold (at 60 min), urinary phosphate rose by a factor of 2 (at 120 min), urine calcium levels fell by 50% between 60 and 120 min, and plasma prolactin rose by a factor of 1.4 (at 60 min). The cAMP responses were significantly blunted in five patients with chronic hypocalcemia, chronic hyperphosphatemia, and detectable serum immunoreactive PTH levels. On the basis of this test these patients were designated as suffering from pseudohypoparathyroidism. The acute phosphaturic and hypocalciuric responses were apparently intact in these five individuals. Human PTH 1-34 is likely to replace bovine material in the delineation of syndromes associated with PTH resistance.

Humoral hypercalcaemia of malignancy: report of two further patients with biochemical studies on tumour extracts.

Tumour extracts from two patients with humoral hypercalcaemia of malignancy contained material which stimulated adenylate cyclase in chick renal membranes and in rat osteosarcoma cells. Adenylate cyclase-stimulating activity in each system was inhibited by a specific parathyroid hormone (PTH) antagonist. Studies in two HPLC systems suggested that the adenylate cyclase-stimulating factors extracted from these tumours differed from each other and from synthetic human parathyroid hormone 1-34. The presence of similar PTH-like adenylate cyclase stimulating material(s) in oncogenic osteomalacia suggests that adenylate cyclase stimulating factor(s) may not be the direct or the sole cause of hypercalcaemia.

Hypervitaminosis A and calcium-regulating hormones in the rat.

The effect of vitamin A on calcium-regulating hormones was studied in rats. A single oral dose of 30 mg retinol equivalents (RE) given to adult rats caused no change to serum biologically active parathyroid hormone (bioactive-PTH) concentrations. Bioactive-PTH secretion from rat thyroparathyroid gland complexes was not significantly altered after in vitro incubation with 1.18 X 10(-6) M retinol. Chronically intoxicated rats given 15 mg RE 3 times a week for 6 wk, showed higher osteoclast numbers and lower osteoid than controls. Serum bioactive-PTH was not detectable and serum 25-hydroxyvitamin D (25-OHD) (25.2 +/- 12.5 nmol/L) was significantly (P less than 0.03) lower than controls (43.3 +/- 3.1). In acutely intoxicated rats (60 mg RE/d for 2 d), serum bioactive-PTH levels were significantly lower (0.02 +/- 0.05 ng/ml, P less than 0.03) than in control animals (0.14 +/- 0.08). Lower doses of vitamin A, 7.5 mg RE 3 times a week for 3 wk, suppressed serum bioactive-PTH to undetectable levels but had no significant effect on serum 25-OHD. Serum calcium and 25-OHD levels were significantly lower in vitamin D-intoxicated rats given 7.5 mg RE 3 times a week (ca. 3.16 +/- 0.19 mmol/L; 25-OHD 599.7 +/- 110.6 nmol/L) than vitamin D-intoxicated controls (3.42 +/- 0.17; 789.3 +/- 17.7). These results suggest that hypervitaminosis A can alter the metabolism of calcium-regulating hormones.

Parathyroid hormone-like bioactivity in tumours from patients with oncogenic osteomalacia.

Extracts from the tumours of three patients with oncogenic osteomalacia stimulated renal adenylate cyclase in a parathyroid hormone (PTH) responsive system. The stimulation was suppressed by a specific PTH antagonist. These observations suggest that osteomalacia-producing tumours exert some of their effects through PTH-like material acting on PTH receptors.

Dialysis osteodystrophy. A study involving 94 patients.

Dynamic skeletal histomorphometry was performed in 94 unselected patients receiving maintenance dialysis for chronic renal failure. An attempt was made to correlate the results with the clinical, biochemical and radiological findings. Skeletal histology was abnormal in each case. Hyperparathyroidism was present as the only abnormality in 18 patients and osteomalacia in 26; 50 patients showed both abnormalities. Osteomalacia, in contrast to hyperparathyroidism, increased in prevalence and severity with the duration of dialysis and with bone aluminum content. The majority of patients had histological osteosclerosis. It was impossible to predict either the nature or the severity of the histological lesions on the basis of symptoms and physical signs or on the basis of most biochemical parameters (including serum concentrations of three vitamin D metabolites). Serum alkaline phosphatase values and serum immunoreactive parathyroid hormone (iPTH) concentrations were positively correlated with the severity of histological hyperparathyroidism. Subperiosteal erosions of the phalanges were associated with severe histological hyperparathyroidism in each case but this radiological sign was absent in 66% of patients with histological hyperparathyroidism. Radiological osteosclerosis was associated with severe histological osteomalacia in each case, but this radiological sign was absent in 87% of patients with histological osteomalacia. No other radiological sign proved a reliable guide to the underlying skeletal histology. In the majority of dialysis patients, a skeletal biopsy is required for an accurate diagnosis of the nature and severity of azotemic osteodystrophy.