Magne K. Fagerhol

Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin

The calcium binding L1 protein was found to inhibit growth of blood culture isolates of Candida spp and cerebrospinal fluid isolates of Cryptococcus neoformans. Minimum inhibitory concentrations (MIC) were 4-128 mg/l, and concentrations 2-4 times the MIC were fungicidal. Blood culture isolates of Escherichia coli, Klebsiella spp, Staphylococcus aureus, and Staphylococcus epidermidis had MIC values of 64-256 mg/l. Antibacterial activity was strongly influenced by the nature of the culture medium. In view of the biological activity of L1, the name calprotectin is proposed to describe this antimicrobial protein with calcium binding properties.

Reduced complement and granulocyte activation with heparin-coated cardiopulmonary bypass.

Plasma concentrations of the complement activation products C3b, iC3b, and C3c; the terminal C5b-9 complement complex; and the granulocyte proteins calprotectin, myeloperoxidase, and lactoferrin were assessed in two groups of patients undergoing aortocoronary bypass procedures. In 10 patients operated on, the bypass circuits were coated by the Carmeda Bio-Active Surface and systemic heparinization was reduced to 1.5 mg/kg; in another 10, the systems were uncoated and the dosage of systemic heparinization was 4 mg/kg. In both groups, significant complement activation was observed after the onset of cardiopulmonary bypass, but the maximum levels of C3b, iC3b, and C3c and the terminal C5b-9 complement complex were significantly lower in the heparin-coated group. In both groups, a significant increase in calprotectin, myeloperoxidase, and lactoferrin release was observed by the end of operation. The maximum myeloperoxidase levels were significantly lower in the heparin-coated group than those in the uncoated group (p = 0.03). There was a correlation of borderline significance between the formation of terminal C5b-9 complement complex and lactoferrin release, as well as between the formation of terminal C5b-9 complement complex and myeloperoxidase release (p = 0.05). The postoperative blood loss did not differ significantly between the two groups. We conclude that coating by end point-attached and functionally active heparin allows a significant reduction in the amount of systemic heparinization, and significantly reduces complement and granulocyte activation.

Calprotectin, a faecal marker of organic gastrointestinal abnormality

Calprotectin may therefore exert its effects by reducing local concentrations of zinc, which indicates that it may be involved in the regulation of inflammatory reactions by its inhibition of several zinc-dependent metalloproteinases. The protein is remarkably resistant to degradation in vivo and in vitro in the presence of calcium, 5,6 so faecal samples can be sent to the laboratory by post. The upper reference limit in faeces is 10 mg/L, but with a recent refinement of the assay, which does away with the messy step of homogenisation and which increases yields, the limit is 50 mg/L. 7 Several groups have confirmed the observation 5 that calprotectin can be assayed in simple buffer extracts of small (5 g) faecal samples, and that increased concentrations (>10 mg/L) are found in most patients with inflammatory bowel disease (IBD) or gastric or colorectal cancer (panel). It was therefore quite clear at an early stage that the faecal calprotectin test was nonspecific for type of organic bowel disease. In different studies the median values in patients with colorectal cancer ranged from 33 to 214 mg/L. In one study 23 patients were also tested after resection, and the median dropped from 75 to 9 mg/L. 8

Plasma levels of the leucocyte L1 protein in febrile conditions: relation to aetiology, number of leucocytes in blood, blood sedimentation reaction and C-reactive protein.

Plasma levels of a novel leucocyte protein (L1) were determined in a series of 176 febrile patients and compared with CRP, BLC, and BSR. Among 82 patients with bacterial infection, 81 had L1 levels above the 0.95 interval. Levels of 40 to 130 times the normal mean were seen frequently during life-threatening infections like septicaemia, meningitis, or pneumonia. In contrast, 23 of 27 patients with viral infections had normal L1 levels. A poor correlation was found between L1 levels, CRP, BLC, and BSR, suggesting that these parameters reflect different aspects of the response to tissue damage and inflammation. L1 and CRP seem equal in distinguishing between bacterial and viral infections. Low or normal L1 levels argue strongly against a bacterial infection, while elevated L1 levels discriminate poorly between bacterial and non-infectious inflammatory or malignant diseases.

Epidermal and dermal distribution of a myelomonocytic antigen (L1) shared by epithelial cells in various inflammatory skin diseases

The L1 antigen is a major cytosol component of human granulocytes that may also be expressed by macrophages and epithelial cells. Its epidermal and dermal occurrence was investigated in formalin-fixed routine biopsy material from eleven different inflammatory skin disorders. Localization was performed with a rabbit antiserum to L1 applied in an unlabeled antibody peroxidase-antiperoxidase method. L1 antigen was not found in normal skin except in epithelial cells of pilosebaceous units. However, epidermal L1 antigen was demonstrated in every biopsy specimen from lupus erythematosus, lichen planus, dermatitis herpetiformis, and atopic dermatitis, whereas granuloma annulare test results were usually negative. The occurrence of dermal L1 antigen depended on the composition of the inflammatory infiltrate; specimens rich in neutrophilic granulocytes (e.g., dermatitis herpetiformis) were particularly strongly stained. Extracellular dermal staining was also seen, especially in areas adjacent to accumulation of positive leukocytes. The varying epidermal occurrence of L1 antigen in skin diseases probably signified different degrees of proliferative activity of the epithelial cells and could apparently not be ascribed to uptake from the dermis.

Antithrombin-III concentration and ABO blood-groups.

An association between blood-group A and an increased risk of thromboembolic disease has been suggested. During blood coagulation antithrombin III (At-III) is partly consumed and the At-III level is higher in the plasma than in the serum. Pulmonary embolism diffuse intravascular coagulation and the use of oral contraceptives are associated with lowered levels of At-III. Normalization of the At-III in response to anticoagulant therapy suggests that the low levels in thromboembolic disease may be due to increased consumption of At-III by complex formation of the thrombin and activated factor x. If individuals of blood group A more often have thrombosis caused by activation of larger amounts of coagulation factors (and/or higher frequency of events) than those of type 0 this might be reflected in lower At-III levels. Such a correlation was found with ABO groups. The mean At-III concentration in blood group A-1 donors was significantly lower than in the other blood groups including A-2 donors. Even when blood groups A-1 and A-2 are pooled the mean value is lower than in group 0. It was concluded that At-III determination is a sensitive method for estimation of intravascular coagulation.

EFFECTS OF HUMAN CALPROTECTIN (L1) ON IN VITRO IMMUNOGLOBULIN SYNTHESIS

Calprotectin (LI) is a major cytoplasmic protein of neutrophilic granulocytes and monocytes/macrophages which is released from leucocytes during activation or cell death. Apart from in vitro antimicrobial and antiproliferative activity little is known about the biological function of the protein. Since previous investigations have shown that Calprotectin plasma levels are elevated in various inflammatory rheumatic diseases, we wanted to investigate if Calprotectin has an effect on immune cell functions. Peripheral blood mononuclear cells, either unstimulated or polyclonally stimulated with mitogen, were incubated with Calprotectin and effects were assessed by enumeration of immunoglobulin secreting cells (ELISPOT).

Effects of calprotectin in avridine‐induced arthritis

Plasma levels of calprotectin correlate with disease activity and clinical assessments of arthritis in various rheumatic diseases, and high levels have been demonstrated in the synovial fluid of patients with rheumatoid arthritis. However, the role of calprotectin in rheumatic inflammation is unclear. The purpose of the present study was to investigate potential intra‐articular effects of calprotectin. Calprotectin was injected into joints of healthy male Lewis rats and into joints of rats in the latency period before onset of avridine‐induced arthritis. In addition, a group of animals had IgG antibodies to rat calprotectin injected into joints before onset of avridine‐induced arthritis. Injection of 0.2 or 10 μ calprotectin into the ankles of healthy male Lewis rats resulted in histologically minor and reversible inflammatory changes, but without any circulating antibodies to calprotectin. Furthermore, animals with 40 μg calprotectin injected into ankles before the expected onset of avridine‐induced arthritis had lower scores for cellular infiltration than were seen in control joints. This difference did not quite reach statistical significance in the two‐sided test used. However, the induced arthritis increased in joints injected with IgG antibodies to calprotectin. These findings may indicate that increased local concentrations of calprotectin are partially protective against avridine‐induced arthritis. In contrast, reduced local concentrations appear to exacerbate the severity of arthritis. Calprotectin may thus be involved in the regulation of inflammatory processes in joints.

Roller and centrifugal pumps compared in vitro with regard to haemolysis, granulocyte and complement activation

A Biomedicus centrifugal pump and a Polystan roller pump were compared in vitro with regard to differences in haemolysis, granulocyte and complement activation. Six circuits of tubing and oxygenators were connected to each pump. Heparinized fresh human blood was circulated for 72 hours in the systems. Blood samples were drawn at defined intervals. Haemolysis was assessed by determination of lactate dehydrogenase (LD) and potassium, and granulocyte activation by quantification of the granulocyte proteins calprotectin, lactoferrin and myeloperoxidase. Complement activation was assessed by measuring C3 activation products (C3b, iC3b and C3c), and the terminal C5b-9 complement complex (TCC). The results indicate more haemolysis and complement activation in the roller pump group, revealed by significantly higher concentrations of LD, potassium, C3 activation products and TCC. Calprotectin, lactoferrin and myeloperoxidase were all significantly increased in both groups, but the rise appeared earlier in the roller pump group. The concentrations of LD and potassium both correlated significantly with C3 activation products, indicating that complement activation may at least partly be responsible for the haemolysis.

L1, a major granulocyte protein; isolation of high quantities of its subunits

L1 is a major granulocyte and monocyte protein with a Mr of 36.5 kDa. It is found mainly in the cytosol of these cells. Purified L1 is shown, on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), to contain three subunits. In this study, 6 mol/l concentration of urea was found to be sufficient for disassembly of the polypeptides, and urea-containing preparative isoelectric focusing gel was used for separation of high quantities of the subunits. The pI of the eluted subunits were 5.8, 6.1 and 7.1. When tested on 2D-PAGE, the isolated subunits were found at their typical locations. Polyclonal rabbit antibodies were produced against the subunits, and the antisera were, on dot-blot, found to react with the different subunits as well as the purified L1.