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Role of neutrophil kinin in infection
Immunopharmacoloyy ELSEVIER
Immunopbarmacology 33 (1996) 387-390
Role of neutrophil kinin in infection Yugen Naidoo *, Strini Naidoo, Raj Nadar, Kanti Daya Bhoola Dept. of Experimental and Clinical Pharmacology, Medical School, P.O. Box 17039, Congella 4013, UniversiO' of Natal, Natal, South Africa
Keywords: Kinin moiety; Pneumonia; Confocal microscopy
1. Introduction
Kinins are ubiquitous mediators which produce many of the cardinal manifestations of inflammation, and for that reason are an important target for therapeutic intervention (Rodell et al., 1995). Inflammation is the normal biological response to tissue injury and is characterized by the interactive activation of multiple mediators and cell types. Activation of these different components of the inflammatory response results in increased blood flow to the injured area and enhances permeability of blood vessels, allowing the movement of cells, such as polymorphonuclear leukocytes (PMNs), macrophages and lymphocytes, into the injured area. Kinins are capable of producing all of the observed cellular effects including vasodilation, venoconstriction and endothelial cell retraction that result in vascular leak, and pain. In addition, kinins have the capacity to release transmitters from nerve terminals (substance P), stimulate the synthesis of cytokines (interleukin-1, tumour necrosis factor), induce the formation of prostaglandins
Abbreviations: B1, bradykinin receptor; B2, bradykinin receptor; PMN, polymorphonuclear; PBS, phosphate buffered saline: TK, tissue kallikrein; PK, plasma kallikrein; SBK1, mouse antibradykinin antibody; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G * Corresponding author.
and leukotrienes by activating phospholipase A2, and release EDRF (nitric oxide) from endothelial cells (Bhoola et al., 1992). Therefore, these bioreactive peptides are attractive autacoids for the mediation of oedema and pain. We have endeavoured to determine the status of the neutrophil kinin in infectious states, and to determine whether in inflammation in which bacterial and cellular proteases are released, the kinin moiety is indeed cleaved from the kininogen molecule on the surface of circulating neutrophils. Previously we reported loss of the kinin moiety from kininogen attached to the external surface of the neutrophil in patients with tuberculosis meningitis (Naidoo et al., 1994). In patients with pneumonia, we extend the hypothesis, that circulating neutrophils show a loss of the kinin moiety in infections.
2. Materials and methods
2.1. Neutrophil isolation Whole blood (10 ml) was obtained by venipuncture through a precision glide vacutainer needle (Becton Dickinson Vacutainer Systems, UK) from healthy volunteers and patients presenting with pneumonia from King Edward VIII Hospital, Durban, Natal. Each 3 ml was drawn into a vacutest tube
0162-3109/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S01 62-3 1 0 9 ( 9 6 ) 0 0 0 9 3 - 8
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Y. Naidoo et aL / Immunopharmacology 33 (1996) 387-390
(Radem Medical, Sandton, SA) containing 0.5 ml of 3.8% (w/v) sodium citrate. The anticoagulated blood was pooled and mixed with an equal volume of phosphate buffered saline (PBS: 150 mM sodium chloride, 200 mM sodium dihydrogen phosphate and 200 mM disodium hydrogen phosphate, pH 7.2). Neutrophils were isolated by careful layering of blood on 3.5 ml Histopaque 1119/1077 (Sigma, UK), and on centrifugation at 15000 g for 30 min at 20°C formed a band. Neutrophils from this layer were harvested with a pasteur pipette, mixed with an equal volume of PBS, centrifuged (500 g), and the process repeated twice.
3. Immunocytochemistry 3.1. Antibodies Antibody for the mature tissue kallikrein (TK), produced in rabbit using purified recombinant TK was kindly given by Dr. Kemme (Germany). Antibody against the kinin moiety was monoclonal (SBK1 supplied by Michael Webb, Sandoz, UK). The dilution of the primary antibody in each of the immunocytochemical procedure was 1:500 (v/v). The kininogen antibody (I 108, provided by Professor Werne Muller-Esterl, Germany) as well as the plasma kallikrein (PK) antibody were also produced in rabbit.
3.2. Light microscopy The isolated neutrophils were pipetted onto a glass slide, air-dried and fixed with paraformaldehyde (2% in PBS). The slides were stored at 4°C until immunostainedo by the peroxidase anti-peroxidase (PAP) method, using the universal PAP kit (Signet Laboratories, Dedham, Massachusetts 02026, USA). The neutrophil smears were sequentially incubated with the appropriate primary antibody (1:500) for 18 h, a sheep anti-rabbit immunoglobulin linking agent for 20 min, and PAP for 40 min. Visualisation of the immhnoenzyme complex was achieved by incubation with 3-amino-9-ethyl-carbazole (AEC, in 1% hydrogen peroxide and 0.1 M acetate buffer, pH 5). After immunostaining, the smears were counterstained with 2% toludine blue, mounted in Kaiser's
jelly, and then viewed under a Nikon Optiphot photomicrocope.
3.3. Confocal microscopy The isolated neutrophils were pipetted onto a glass slide, air-dried and fixed with paraformaldehyde (4% in PBS). The slides were then stored at 4°C until viewed by confocal microscope (Leica, Heidelberg, Germany). The neutrophil smears were first rinsed with PBS containing bovine serum albumin (1% v/v) for 20 rain. Next, the cells were sequentially incubated with the appropriate primary antibody (1:500 for 3 h), rinsed in PBS, and thereafter incubated with fluorescein-isothiocyanate (FITC)-conjugated F(ab) 2 fragments of antispecies IgG (1 : 250, v/v) for 30 min. The specific fluorescence of the immunostained enzyme within cells was viewed with a confocal microscope equipped with an excitatory argon laser.
3.4. Electron microscopy The isolated neutrophils were washed with PBS containing sodium azide (0.2%). The suspended cells were pipetted into Eppendorf tubes and then sequentially the cells were prefixed with paraformaldehyde (1% in PBS) for 15 min, incubated with the primary antibody (1 : 500, v/v) for 3 h, incubated with Auroprobe EM reagent coupled with 10 nm gold particles for 2 h (Amersham International, Buckinghamshire, UK), fixed overnight in gluteraldehyde (4% in PBS); between each of the steps, the cells were washed with PBS containing bovine serum albumin (1%, v/v) and human IgG (1%, v/v), and centrifuged at 300 g for 4 min. After the final fixation step, the cells were embedded in epoxy-araldite resin. Ultrathin sections were cut on a Reichert Ultracut ultramicrotome, counterstained with lead citrate and viewed in a Joel 100C electron microscope.
3.5. Immunocytochemical controls The immunolocalisation controls included omission of the primary antibody or replacement by non-immune serum. Controls also included preabsorption of the diluted, specific antibody with the respective antigen. For immunoelectron microscopy,
E Naidoo et al. / Immunopharmacology 33 (1996) 387-390
Fig.1
Fig.2
Fig.3
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390
Y. Naidoo et al. / Immunopharmacology 33 (1996) 387-390
omission or replacement of the specific antiserum resulted in absence of specific gold labelling on the surface of the cell.
4. Results The monoclonal antibody, SBK1, immunoreacted intensely with the in situ neutrophil kinin in control blood when viewed by conventional light microscopy (Fig. la). However, in pneumonia patients, no reaction product was observed on the neutrophils (Fig. lb). However, the presence of kininogen as well as PK on the neutrophil surface was unaffected in these patients. Using confocal microscopy, the image processing programmes recorded the fluorescence emitted from the FITC-labelled F(ab)2 secondary antibody, linked to the specific primary antibody, with a pseudocolour gradient that ranged from white-red (maximal) to blue (nil), giving an estimate of the amount of antigen present in each optical plane. There was a distinct loss of the kinin moiety on the surface of neutrophils harvested from pneumonic patients (Fig. 2a), whereas the kinin segment remained unaltered in neutrophils from control blood cells (Fig. 2b). Confirmation electron microscopy studies showed immunoreactive kinin as clusters of gold deposits on the surface of the neutrophil from control blood (Fig. 3a), whereas no gold particles were seen in sections prepared from pneumonia individuals (Fig. 3b).
loss of the kinin moiety on the surface of the neutrophil in patients presenting with pneumonia. The confocal images in fixed, permeabilised neutrophils provided multi-dimensional constructs of the kinin molecule, and the intensity of fluorescence reflected the relative amounts of the molecule present in both neutrophils harvested from healthy as well as pneumonic patients. The immunogold experiments confirmed, at the ultrastructural level, the presence or disappearance of the kinin moiety from the kininogen molecule on the neutrophil surface. This finding confirms our hypothesis that there is a loss of the kinin moiety from circulatory neutrophils in systemic infections. Neutrophils chemotactically drawn to the site of inflammation are activated to release the kinin segment from the kininogen molecule, thereafter reenter the circulation, and therefore can be harvested. This discovery could prove useful in drug therapy of organ and systemic infections.
References Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens and kininase. Pharmacol Rev. 1992; 44: 1-80. Rodell TC, Naidoo Y, Bhoola KD. Role of kinins in inflammatory responses: Prospectsfor drug therapy. Clin Immunother1995; 3(5):352-361. Naidoo Y, Snyman C, Narotam PK, Bhoola KD. Can J Phys Pharm 1994; 72(2): 40.
5. Discussion Our immunocytochemical study with the monoclonal anti-kinin antibody clearly demonstrated a
Fig. 1. Light microscopy. (a) ImmunoreactiveSBK1 on the neutrophil surface of healthy volunteers. (b) Loss of the kinin moiety in pneumonic patients. Fig. 2. Confocalmicroscopy.(a) Loss of the kinin moietyon the neutrophil surface of pneumonicpatients. (b) Immunreactivekinin on the neutrophil surface of normal blood. Fig. 3. Electron microscopy.(a) Normalneutrophil showing kinin as clusters of gold deposits. (b) Absence of immuffostainingfor kinin in pneumonic patients.
Immunopbarmacology 33 (1996) 387-390
Role of neutrophil kinin in infection Yugen Naidoo *, Strini Naidoo, Raj Nadar, Kanti Daya Bhoola Dept. of Experimental and Clinical Pharmacology, Medical School, P.O. Box 17039, Congella 4013, UniversiO' of Natal, Natal, South Africa
Keywords: Kinin moiety; Pneumonia; Confocal microscopy
1. Introduction
Kinins are ubiquitous mediators which produce many of the cardinal manifestations of inflammation, and for that reason are an important target for therapeutic intervention (Rodell et al., 1995). Inflammation is the normal biological response to tissue injury and is characterized by the interactive activation of multiple mediators and cell types. Activation of these different components of the inflammatory response results in increased blood flow to the injured area and enhances permeability of blood vessels, allowing the movement of cells, such as polymorphonuclear leukocytes (PMNs), macrophages and lymphocytes, into the injured area. Kinins are capable of producing all of the observed cellular effects including vasodilation, venoconstriction and endothelial cell retraction that result in vascular leak, and pain. In addition, kinins have the capacity to release transmitters from nerve terminals (substance P), stimulate the synthesis of cytokines (interleukin-1, tumour necrosis factor), induce the formation of prostaglandins
Abbreviations: B1, bradykinin receptor; B2, bradykinin receptor; PMN, polymorphonuclear; PBS, phosphate buffered saline: TK, tissue kallikrein; PK, plasma kallikrein; SBK1, mouse antibradykinin antibody; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G * Corresponding author.
and leukotrienes by activating phospholipase A2, and release EDRF (nitric oxide) from endothelial cells (Bhoola et al., 1992). Therefore, these bioreactive peptides are attractive autacoids for the mediation of oedema and pain. We have endeavoured to determine the status of the neutrophil kinin in infectious states, and to determine whether in inflammation in which bacterial and cellular proteases are released, the kinin moiety is indeed cleaved from the kininogen molecule on the surface of circulating neutrophils. Previously we reported loss of the kinin moiety from kininogen attached to the external surface of the neutrophil in patients with tuberculosis meningitis (Naidoo et al., 1994). In patients with pneumonia, we extend the hypothesis, that circulating neutrophils show a loss of the kinin moiety in infections.
2. Materials and methods
2.1. Neutrophil isolation Whole blood (10 ml) was obtained by venipuncture through a precision glide vacutainer needle (Becton Dickinson Vacutainer Systems, UK) from healthy volunteers and patients presenting with pneumonia from King Edward VIII Hospital, Durban, Natal. Each 3 ml was drawn into a vacutest tube
0162-3109/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S01 62-3 1 0 9 ( 9 6 ) 0 0 0 9 3 - 8
388
Y. Naidoo et aL / Immunopharmacology 33 (1996) 387-390
(Radem Medical, Sandton, SA) containing 0.5 ml of 3.8% (w/v) sodium citrate. The anticoagulated blood was pooled and mixed with an equal volume of phosphate buffered saline (PBS: 150 mM sodium chloride, 200 mM sodium dihydrogen phosphate and 200 mM disodium hydrogen phosphate, pH 7.2). Neutrophils were isolated by careful layering of blood on 3.5 ml Histopaque 1119/1077 (Sigma, UK), and on centrifugation at 15000 g for 30 min at 20°C formed a band. Neutrophils from this layer were harvested with a pasteur pipette, mixed with an equal volume of PBS, centrifuged (500 g), and the process repeated twice.
3. Immunocytochemistry 3.1. Antibodies Antibody for the mature tissue kallikrein (TK), produced in rabbit using purified recombinant TK was kindly given by Dr. Kemme (Germany). Antibody against the kinin moiety was monoclonal (SBK1 supplied by Michael Webb, Sandoz, UK). The dilution of the primary antibody in each of the immunocytochemical procedure was 1:500 (v/v). The kininogen antibody (I 108, provided by Professor Werne Muller-Esterl, Germany) as well as the plasma kallikrein (PK) antibody were also produced in rabbit.
3.2. Light microscopy The isolated neutrophils were pipetted onto a glass slide, air-dried and fixed with paraformaldehyde (2% in PBS). The slides were stored at 4°C until immunostainedo by the peroxidase anti-peroxidase (PAP) method, using the universal PAP kit (Signet Laboratories, Dedham, Massachusetts 02026, USA). The neutrophil smears were sequentially incubated with the appropriate primary antibody (1:500) for 18 h, a sheep anti-rabbit immunoglobulin linking agent for 20 min, and PAP for 40 min. Visualisation of the immhnoenzyme complex was achieved by incubation with 3-amino-9-ethyl-carbazole (AEC, in 1% hydrogen peroxide and 0.1 M acetate buffer, pH 5). After immunostaining, the smears were counterstained with 2% toludine blue, mounted in Kaiser's
jelly, and then viewed under a Nikon Optiphot photomicrocope.
3.3. Confocal microscopy The isolated neutrophils were pipetted onto a glass slide, air-dried and fixed with paraformaldehyde (4% in PBS). The slides were then stored at 4°C until viewed by confocal microscope (Leica, Heidelberg, Germany). The neutrophil smears were first rinsed with PBS containing bovine serum albumin (1% v/v) for 20 rain. Next, the cells were sequentially incubated with the appropriate primary antibody (1:500 for 3 h), rinsed in PBS, and thereafter incubated with fluorescein-isothiocyanate (FITC)-conjugated F(ab) 2 fragments of antispecies IgG (1 : 250, v/v) for 30 min. The specific fluorescence of the immunostained enzyme within cells was viewed with a confocal microscope equipped with an excitatory argon laser.
3.4. Electron microscopy The isolated neutrophils were washed with PBS containing sodium azide (0.2%). The suspended cells were pipetted into Eppendorf tubes and then sequentially the cells were prefixed with paraformaldehyde (1% in PBS) for 15 min, incubated with the primary antibody (1 : 500, v/v) for 3 h, incubated with Auroprobe EM reagent coupled with 10 nm gold particles for 2 h (Amersham International, Buckinghamshire, UK), fixed overnight in gluteraldehyde (4% in PBS); between each of the steps, the cells were washed with PBS containing bovine serum albumin (1%, v/v) and human IgG (1%, v/v), and centrifuged at 300 g for 4 min. After the final fixation step, the cells were embedded in epoxy-araldite resin. Ultrathin sections were cut on a Reichert Ultracut ultramicrotome, counterstained with lead citrate and viewed in a Joel 100C electron microscope.
3.5. Immunocytochemical controls The immunolocalisation controls included omission of the primary antibody or replacement by non-immune serum. Controls also included preabsorption of the diluted, specific antibody with the respective antigen. For immunoelectron microscopy,
E Naidoo et al. / Immunopharmacology 33 (1996) 387-390
Fig.1
Fig.2
Fig.3
389
390
Y. Naidoo et al. / Immunopharmacology 33 (1996) 387-390
omission or replacement of the specific antiserum resulted in absence of specific gold labelling on the surface of the cell.
4. Results The monoclonal antibody, SBK1, immunoreacted intensely with the in situ neutrophil kinin in control blood when viewed by conventional light microscopy (Fig. la). However, in pneumonia patients, no reaction product was observed on the neutrophils (Fig. lb). However, the presence of kininogen as well as PK on the neutrophil surface was unaffected in these patients. Using confocal microscopy, the image processing programmes recorded the fluorescence emitted from the FITC-labelled F(ab)2 secondary antibody, linked to the specific primary antibody, with a pseudocolour gradient that ranged from white-red (maximal) to blue (nil), giving an estimate of the amount of antigen present in each optical plane. There was a distinct loss of the kinin moiety on the surface of neutrophils harvested from pneumonic patients (Fig. 2a), whereas the kinin segment remained unaltered in neutrophils from control blood cells (Fig. 2b). Confirmation electron microscopy studies showed immunoreactive kinin as clusters of gold deposits on the surface of the neutrophil from control blood (Fig. 3a), whereas no gold particles were seen in sections prepared from pneumonia individuals (Fig. 3b).
loss of the kinin moiety on the surface of the neutrophil in patients presenting with pneumonia. The confocal images in fixed, permeabilised neutrophils provided multi-dimensional constructs of the kinin molecule, and the intensity of fluorescence reflected the relative amounts of the molecule present in both neutrophils harvested from healthy as well as pneumonic patients. The immunogold experiments confirmed, at the ultrastructural level, the presence or disappearance of the kinin moiety from the kininogen molecule on the neutrophil surface. This finding confirms our hypothesis that there is a loss of the kinin moiety from circulatory neutrophils in systemic infections. Neutrophils chemotactically drawn to the site of inflammation are activated to release the kinin segment from the kininogen molecule, thereafter reenter the circulation, and therefore can be harvested. This discovery could prove useful in drug therapy of organ and systemic infections.
References Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens and kininase. Pharmacol Rev. 1992; 44: 1-80. Rodell TC, Naidoo Y, Bhoola KD. Role of kinins in inflammatory responses: Prospectsfor drug therapy. Clin Immunother1995; 3(5):352-361. Naidoo Y, Snyman C, Narotam PK, Bhoola KD. Can J Phys Pharm 1994; 72(2): 40.
5. Discussion Our immunocytochemical study with the monoclonal anti-kinin antibody clearly demonstrated a
Fig. 1. Light microscopy. (a) ImmunoreactiveSBK1 on the neutrophil surface of healthy volunteers. (b) Loss of the kinin moiety in pneumonic patients. Fig. 2. Confocalmicroscopy.(a) Loss of the kinin moietyon the neutrophil surface of pneumonicpatients. (b) Immunreactivekinin on the neutrophil surface of normal blood. Fig. 3. Electron microscopy.(a) Normalneutrophil showing kinin as clusters of gold deposits. (b) Absence of immuffostainingfor kinin in pneumonic patients.