Activation of human neutrophils by monoclonal antibody PMN7C3: cell movement and adhesion can be triggered independently from the respiratory burst

Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.

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Activation of human neutrophils by monoclonal antibody PMN7C3: cell movement and adhesion can be triggered independently from the respiratory burst

Blood ◽

10.1182/blood.v67.5.1388.1388 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1388-1394 ◽

Cited By ~ 11

Author(s):

DA Melnick ◽

T Meshulam ◽

A Manto ◽

HL Malech

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Respiratory Burst ◽

Cellular Response ◽

Rapid Change ◽

Cell Movement ◽

Human Neutrophils ◽

Free Calcium ◽

Transient Rise ◽

Free Calcium Concentration

Abstract Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.

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IgM monoclonal antibodies recognizing FcαR but not FcγRIII trigger a respiratory burst in neutrophils although both trigger an increase in intracellular calcium levels and degranulation

Biochemical Journal ◽

10.1042/bj3060519 ◽

1995 ◽

Vol 306 (2) ◽

pp. 519-523 ◽

Cited By ~ 15

Author(s):

S J Mackenzie ◽

M A Kerr

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Intracellular Calcium ◽

Respiratory Burst ◽

Calcium Concentration ◽

Intracellular Calcium Concentration ◽

Human Neutrophils ◽

Enhanced Chemiluminescence ◽

Cell Surface Carbohydrate ◽

Small Rise

The role of IgG receptor, Fc gamma RIII, in the triggering of neutrophil functions has been controversial. Here we show that IgM monoclonal antibodies, Leu 11b and 1D3, recognizing Fc gamma RIII, bind to human neutrophils triggering an increase in intracellular calcium concentration and release of myeloperoxidase upon degranulation but do not trigger a respiratory burst detectable as lucigenin-enhanced chemiluminescence. Although many fewer molecules of IgM monoclonal antibody, MY43, recognizing Fc alpha R, bind to the same cells they trigger a much greater increase in intracellular calcium concentration, release of myeloperoxidase and a strong respiratory burst. Since the respiratory bursts triggered by IgG and IgA are equivalent, this demonstrates that Fc gamma RII is responsible for the IgG-mediated response. IgM monoclonal antibody MC2, recognizing the abundant neutrophil cell-surface carbohydrate CD15, also triggers a small rise in intracellular calcium but no respiratory burst.

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Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease

Blood ◽

10.1182/blood.v63.1.231.231 ◽

1984 ◽

Vol 63 (1) ◽

pp. 231-233 ◽

Cited By ~ 20

Author(s):

PD Lew ◽

C Wollheim ◽

RA Seger ◽

T Pozzan

Keyword(s):

Chronic Granulomatous Disease ◽

Calcium Concentration ◽

Cytosolic Calcium ◽

Human Neutrophils ◽

Free Calcium ◽

Granulomatous Disease ◽

Chemotactic Peptide ◽

Intact Cells ◽

Calcium Indicator ◽

Free Calcium Concentration

Abstract Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.

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Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes

Blood ◽

10.1182/blood.v78.4.1112.1112 ◽

1991 ◽

Vol 78 (4) ◽

pp. 1112-1116 ◽

Cited By ~ 239

Author(s):

BJ Rollins ◽

A Walz ◽

M Baggiolini

Keyword(s):

Growth Factor ◽

Respiratory Burst ◽

Specific Activity ◽

Calcium Flux ◽

Human Neutrophils ◽

Free Calcium ◽

Secretory Proteins ◽

Predicted Amino Acid Sequence ◽

Human Monocytes ◽

Inducible Gene

Abstract The JE gene was first described as a platelet-derived growth factor (PDGF)-inducible gene in mouse 3T3 cells. The human homologue of JE encodes a protein whose predicted amino acid sequence is identical to that of the monocyte chemoattractant MCP-1 (also called MCAF and SMC- CF), which belongs to a recently identified family of small secretory proteins with cytokine properties. We purified recombinant human MCP- 1/JE (hMCP-1/JE) produced in COS cells and demonstrated that it is chemotactic for human monocytes with a specific activity similar to natural MCP-1. In addition, pure recombinant hMCP-1/JE stimulates monocytes, inducing an increase in cytosolic free calcium and the respiratory burst, but is completely inactive on human neutrophils. These results help to define functionally a well-known growth factor- inducible gene and a member of a new family of cytokines.

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The effect of mitogenic lectins and monoclonal antibodies on intracellular free calcium concentration in human T-lymphocytes

Biochemical Journal ◽

10.1042/bj2190661 ◽

1984 ◽

Vol 219 (2) ◽

pp. 661-666 ◽

Cited By ~ 59

Author(s):

K O'Flynn ◽

D C Linch ◽

P E R Tatham

Keyword(s):

T Cells ◽

Monoclonal Antibodies ◽

T Cell ◽

Calcium Concentration ◽

Human Lymphocytes ◽

Maximum Level ◽

T Cell Response ◽

Free Calcium ◽

Intracellular Free Calcium Concentration ◽

Free Calcium Concentration

The effect of mitogenic lectins and the mitogenic antibody UCHT1 on the cytoplasmic free Ca2+ concentration ([Ca2+]i) in human lymphocytes was investigated by using the fluorescent Ca2+-indicator quin2 . Phytohaemagglutinin, concanavalin A and UCHT1 increased [Ca2+]i in T-cells to a maximum level within 2 min. No T-cell response was seen with poke weed mitogen. None of the mitogens affected non-T-cell [Ca2+]i.

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Attachment to fibronectin or vitronectin makes human neutrophil migration sensitive to alterations in cytosolic free calcium concentration.

The Journal of Cell Biology ◽

10.1083/jcb.112.1.149 ◽

1991 ◽

Vol 112 (1) ◽

pp. 149-158 ◽

Cited By ~ 66

Author(s):

P W Marks ◽

B Hendey ◽

F R Maxfield

Keyword(s):

Calcium Concentration ◽

Neutrophil Migration ◽

Human Neutrophils ◽

Free Calcium ◽

Cytosolic Free Calcium ◽

Coated Glass ◽

Free Calcium Concentration ◽

Cytosolic Free Calcium Concentration ◽

Migration Of Cells

Transient increases in cytosolic free calcium concentration, [Ca2+]i, appear to be required for the migration of human neutrophils on poly-D-lysine-coated glass in the presence of dilute serum (Marks, P. W., and F. R. Maxfield. 1990. J. Cell Biol. 110:43-52). In contrast, no requirement for [Ca2+]i transients exists when neutrophils migrate on albumin-coated glass in the absence of serum. To determine the mechanism that necessitates [Ca2+]i transients on poly-D-lysine in the presence of serum, migration was examined on substrates consisting of purified adhesive glycoproteins. In the absence of external Ca2+, a treatment which causes the cessation of [Ca2+]i transients, migration on fibronectin (fn) and vitronectin (vn) was significantly inhibited. Migration was also inhibited in Ca2(+)-buffered cells on these substrates, indicating that this effect was the result of an alteration of [Ca2+]i. In the absence of external Ca2+, the inhibition of migration on fn or vn was more pronounced when soluble fn or vn was added to cells migrating on these substrates. This effect of soluble adhesive glycoprotein was specific: in the absence of external Ca2+, soluble fn did not affect the migration of cells on vn, and soluble vn did not affect the migration on fn. No additional inhibition of migration was observed in Ca2(+)-buffered cells with the addition of soluble adhesive glycoprotein. These data indicate that [Ca2+]i transients are involved in continued migration of human neutrophils on fn or vn, proteins which are part of the extracellular matrix that neutrophils encounter in vivo.

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Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease

Blood ◽

10.1182/blood.v63.1.231.bloodjournal631231 ◽

1984 ◽

Vol 63 (1) ◽

pp. 231-233

Author(s):

PD Lew ◽

C Wollheim ◽

RA Seger ◽

T Pozzan

Keyword(s):

Chronic Granulomatous Disease ◽

Calcium Concentration ◽

Cytosolic Calcium ◽

Human Neutrophils ◽

Free Calcium ◽

Granulomatous Disease ◽

Chemotactic Peptide ◽

Intact Cells ◽

Calcium Indicator ◽

Free Calcium Concentration

Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.

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Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells

Physiological Genomics ◽

10.1152/physiolgenomics.2001.5.1.11 ◽

2001 ◽

Vol 5 (1) ◽

pp. 11-19 ◽

Cited By ~ 69

Author(s):

KATHLEEN G. MOUNTJOY ◽

PHILIP L. KONG ◽

JOHN A. TAYLOR ◽

DERRIL H. WILLARD ◽

WILLIAM O. WILKISON

Keyword(s):

Intracellular Free Calcium ◽

Melanocortin Receptor ◽

Hek293 Cells ◽

Melanocortin Receptors ◽

Calcium Signal ◽

Free Calcium ◽

Agouti Protein ◽

Transient Rise ◽

Physiological Relevance ◽

Free Calcium Concentration

Mouse melanocortin receptors, MC1-R, MC3-R, MC4-R, and MC5-R, when expressed in HEK293 cells and stimulated with either α-melanocyte-stimulating hormone (α-MSH) or desacetyl-α-MSH, mediate increases in intracellular free calcium concentration ([Ca2+]i) with EC50 values between 0.3 and 4.3 nM. The increase in [Ca2+]i is cholera toxin sensitive and pertussis toxin insensitive. The mechanism involves calcium mobilization from intracellular stores without a transient rise in inositol trisphosphate. Mouse agouti protein (55 nM) is a competitive antagonist of α-MSH (6-fold) and desacetyl-α-MSH (8-fold), coupling the mMC1-R to increased [Ca2+]i. Agouti protein (55 nM) significantly increased the EC50 for α-MSH (3-fold), and 550 nM agouti protein significantly increased the EC50 for desacetyl-α-MSH (4-fold), coupling the mMC4-R to a rise in [Ca2+]i. However, agouti protein antagonism of the MC4-R may not be competitive since there was a trend for the maximum response to also increase. There was no significant antagonism of the MC3-R and MC5-R by agouti protein (55 nM). Understanding the physiological relevance of the transduction of a calcium signal by melanocortin peptides may be important for future development of therapeutic targeting of the melanocortin receptors.

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