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The binding of human monocyte chemoattractant protein-1 (MCP-1) to human monocytes was studied. MCP-1 was radioiodinated with Iodo-beads (Pierce Chemical Co., Rockford, IL) without significant loss of biologic activity. 125I-MCP-1 binding to PBMC occurred within 5 min at 0 degrees C and the binding was inhibited by unlabeled MCP-1 dose dependently but not by neutrophil attractant/activation protein-1 or FMLP. 125I-MCP-1 bound to monocytes; no significant binding to either neutrophils or lymphocytes was observed. Scatchard plot analysis indicated that monocytes had a minimum of 1700 +/- 600 binding sites per cell with a Kd of 1.9 +/- 0.2 x 10(-9) M. For analysis of binding by flow cytometry, MCP-1 was biotinylated. In contrast to radioiodination, biotinylation resulted in loss of activity; potency was 10-fold less, but the efficacy was retained. Detection by flow cytometry of bound biotinylated MCP-1 with avidin-FITC confirmed results obtained with 125I-MCP-1. Biotinylated MCP-1 bound to monocytes but not to lymphocytes; and the binding was inhibited by a 100-fold excess of unlabeled MCP-1.

Neutrophil attractant/activation protein-1 (NAP-1) was previously shown to attract human neutrophils, but not monocytes. The purpose of this study was to determine if NAP-1 interacted with other types of blood leukocytes. In addition to its chemotactic activity for neutrophils, NAP-1 induced chemotactic responses by T lymphocytes and basophils. Chemotactic potency (10(-8) M for an optimal response) was the same for all three cell types. However, NAP-1 caused a chemotactic response in excess of random migration of 7% or 16% of basophils (depending on the medium used) and only 9% of T lymphocytes, in contrast to 30% of neutrophils. This agonist was not chemotactic for partially purified normal human eosinophils. The symmetrical histogram obtained by flow cytometry of neutrophils equilibrated at 0 degree C with fluoresceinated NAP-1 indicates that all neutrophils bound the ligand. A dose-response curve plateau, and inhibition of binding of NAP-1-FITC by unlabeled ligand are evidence for saturable binding to receptors, estimated to be 7000 per cell. Our results suggest that, for induction of an acute inflammatory response, the quantitatively significant action of NAP-1 is on neutrophils.

Only 20 to 40% of human blood monocytes were capable of responding to chemotaxins in vitro. This limit is not due to restrictions of the in vitro system, but is due to the existence of a migrating subpopulation. Over a wide range, the number of cells migrating toward a given concentration of chemotaxin was directly proportional to the number added to the chemotaxis chamber. These monocytes responded to all of the three stimuli used: human serum-derived C5a, human lymphocyte-derived chemotactic factor, and a synthetic peptide. It was possible to deactivate cells to one attractant, leaving the response to other attractants intact. This suggested that these attractants were recognized by different receptors. Several lines of evidence showed that most migrating cells had receptors for all three chemotaxins tested. Thus, if cells were assayed for migration to one attractant, no additional migration occurred when the remaining cells were assayed for migration to a different attractant. Furthermore, the same cells that had migrated toward one attractant were able to respond to other chemotaxins. We also found that a single attractant attracted as many cells as a combination of two or three attractants. Calculations from these data showed that at least 75% of the migrating monocytes have different receptors for all three attractants.

Human peripheral blood monocytes comprise a subpopulation of 20 to 40% that is capable of responding to chemoattractants and a remaining subpopulation that cannot respond. We were able to obtain 99%-pure attractant-responsive monocytes by using a newly constructed separation chamber. The binding of the radioactive chemotactic peptide N-formylmethionyl-leucyl-[3H]phenylalanine to migrating and nonmigrating populations was then studied. The binding was saturable at room temperature in the presence of azide. Saturation occurred at 5 x 10(-8) M, and 50% of the maximal binding was obtained at 10(-8) M, the concentration that induced optimal chemotaxis. The nonmigrating monocytes did not bind the peptide under the same conditions, which shows that at least one reason for a nonresponsiveness to chemotaxin is apparently a lack of receptors. By Scatchard analysis we calculated an equilibrium dissociation constant ranging from 23 to 37 nM; the number of binding sites per cell ranged from 64,000 to 77,000. The binding was very rapid. Fifty percent of the optimal binding occurred at 3.5 min, and equilibrium was reached after 20 to 30 min. Chemotactic deactivation of the monocytes reduced the number of available binding sites by 60%.

A neutrophil chemotaxis assay was developd which permits rapid, quantitative assessment of migration across a membrane filter. The critical factor in the assay was the use of a 10 microns thick polycarbonate membrane without the usual polyvinylpyrrolidone coating. Migrated neutrophils remain adherent to the uncoated membrane, whereas 20-50% fall off polyvinylpyrrolidone-coated membranes. A major advantage of the method is that neutrophil chemotaxis can be readily quantified, since the migrated cells adhere to the membrane surface and are in one optical plane for counting. A 25 mm x 80 mm membrane sheet was used in a 48-well micro chemotaxis assembly, which requires only 20,000 neutrophils and 25 microliters of attractant per assay well. Neutrophil chemotaxis was complete within 10-20 min at 37 degrees C, with 20-30% of the cells migrating to N-formyl-methionyl-leucyl-phenylalanine and 40-50% migrating to complement derived C5a.

Normal human blood contains a population of neutrophils that migrates to various chemoattractants and a population that fails to migrate. The percentage of neutrophils migrating to optimal concentrations of chemoattractants was quantified: 20 to 40% migrated to N-formylmethionyl-leucyl-phenylalanine, 30 to 50% migrated to human C5a, 25 to 35% migrated to human leukocyte-derived chemotactic factors, 20 to 30% migrated to casein, 15 to 20% migrated to pepstatin, and 1 to 5% migrated to medium alone. Neutrophil migration to the most active chemoattractant was not increased when other chemoattractants were added, indicating that the population of neutrophils migrating to the most active attractant was the same population that was migrating to the other attractants. The percentage of neutrophils migrating to a chemoattractant was not altered by prolonging the assay incubation period or by replacing the attractant with new chemoattractant during the assay, and the percentage was independent of the neutrophil concentration added to the chemotaxis chamber. Nonmigrating neutrophils were isolated with a chemotaxis collection chamber, and they were examined for radiolabeled chemotactic peptide binding. The binding of radiolabeled N-formylmethionyl-leucyl-phenylalanine by nonmigrating and migrating neutrophils was identical.

The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein-1 (MCP-1), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5'-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389-nucleotide 3'-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP-1 gene, which is conserved in several primates. MCP-1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL-1, but not by IL-2, TNF, or IFN-gamma. Among proteins with similar sequences, the coding regions of MCP-1 and mouse JE show 68% identity. This suggest that MCP-1 is the human homologue of the mouse competence gene JE.

Although all human neutrophils have receptors for formyl-methionyl-leucyl-phenylalanine, only 20-30% migrate in vitro at optimal attractant concentrations. Since this attractant can be oxidized by myeloperoxidase from stimulated neutrophils, we determined if its efficacy was increased by protection from oxidation. Efficacy was increased by reducing agents and by molecules with oxidizable sulfur, 10(-5) M methionine or 1.5 X 10(-6) M albumin. The antioxidant effect was on the attractant, not the cells: albumin in the cell compartment did not increase responses. Furthermore, antioxidants had no effect on efficacy of fNle-Leu-Phe-Nle-Tyr-Lys, an attractant that also stimulated superoxide release but had no oxidizable sulfur.

Stimulated human monocytes release several proteins thought to play a role in inflammation, including interleukin 1, tumor necrosis factor, and plasminogen activator. We have purified another proinflammatory protein that is chemotactic for human neutrophils from conditioned medium of lipopolysaccharide-stimulated monocytes. After a series of steps that included anion-exchange chromatography, gel filtration, and HPLC on cation-exchange and reverse-phase columns, an apparently pure protein was obtained that migrated as a single 7-kDa band on NaDodSO4/polyacrylamide gels under reducing or nonreducing conditions. The amino acid composition of this monocyte-derived neutrophil chemotactic factor was different from that of interleukin 1 and tumor necrosis factor. N-terminal amino acid sequence of the first 42 residues was determined. This portion of the molecule has up to 56% sequence similarity with several proteins that may be involved in host responses to infection or tissue injury. It is identical to a portion of a sequence deduced from an mRNA induced by staphylococcal enterotoxin treatment of human leukocytes. At the optimal concentration of 10 nM, 50% of neutrophils added to chemotaxis assay wells migrated toward the pure attractant. Potency and efficacy are comparable to that of fMet-Leu-Phe, which is often used as a reference. In contrast to many attractants, the protein was not chemotactic for human monocytes.

Histamine in concentrations as low as 10(-8) M inhibited the chemotactic response of normal human basophils to C5a. Histamine had no effect on basophil chemotaxis to lymphocyte-derived chemotactic factor. Histamine inhibition of basophil chemotaxis to C5a was prevented by metiamide, a drug which blocks H2 receptors for histamine. Since the accumulation of basophils in delayed cutaneous basophilic hypersensitivity (CBH) reactions may occur in part because of chemotaxis to C5a, and since C5a can induce histamine release, histamine inhibition of chemotaxis may limit basophil infiltration in CBH lesions.

The chemotactic deactivation of human monocytes was studied to provide insight into the mechanism of chemotaxis. Deactivation was dependent on the dose of chemoattractant and time of incubation. A concentration in the cell suspension of 10(-8) M N-formylmethionylleucyl phenylalanine (FMLP) for 45 min at 37 degrees C led to 60% suppression of the subsequent specific chemotactic response. Higher concentrations of FMLP led to almost 100% specific suppression. Deactivation was specific under all conditions used. The response to a nonrelated chemoattractant, human serum-derived C5a, was unaffected by incubation in FMLP. Deactivation was also transient. If cells were deactivated at 37 degrees C with FMLP, they recovered within 6 h at 37 degrees C from this deactivation. Both phenomena, deactivation and recovery from deactivation, were temperature dependent. Monocytes could not be deactivated at 0 degrees C, and they did not recover from deactivation when kept at 0 degrees C. Thus, specific deactivation appears to require cellular metabolism, involving loss of receptors or blocking of a step between receptor occupancy and response.

A chemotactic tetrapeptide from culture fluids of Staphylococcus aureus was purified to homogeneity by reverse-phase high-pressure liquid chromatography. The peptide comprises equimolar methionine, leucine, isoleucine, and phenylalanine. It inhibited binding of fluoresceinated fMet-Leu-Phe-Lys to human monocytes, which showed that it interacted with the formyl-methionyl peptide receptor and suggested that it was a formyl-methionyl peptide. Based on a comparison of dose-response curves for inhibition of fluoresceinated fMet-Leu-Phe-Lys binding, the relative affinity of the peptide for the receptor was comparable to that of fMet-Leu-Phe-Lys. At optimal concentrations, chemotactic efficacy (percentage of monocytes migrating to the attractant) was 53 +/- 4%, in contrast to 36 +/- 3% for the reference attractant fMet-Leu-Phe. Since approximately 60% of human monocytes have formyl-peptide receptors, the bacterial peptide is capable of attracting all receptor-bearing monocytes.

The chemotactic peptide fMet-Leu-Phe-Lys is a potent chemoattractant for human blood monocytes. However, only one-third of the monocytes respond. To determine whether or not lack of response reflected absence of attractant receptors, we equilibrated peripheral blood mononuclear cells with fMet-Leu-Phe-Lys-FITC and analyzed binding by flow cytometry. The fluoresceinated peptide bound rapidly at 0 degree C, and the amount bound approached saturation with increasing concentration. The percentage of blood monocytes that bound the peptide was 60 +/- 8 (SEM for seven experiments). In contrast, only 36 +/- 3% (SEM for 16 experiments) of monocytes responded to the attractant by directed migration. It follows that, among the 64 nonmigrating monocytes per 100 total monocytes, approximately 40, or two-thirds of them, fail to bind attractant; the remaining one-third bind attractant but do not respond with directed movement.

MDNCF is a human monocyte-derived, 72-residue chemotactic peptide, which has sequence similarity with members of a family of pro-inflammatory cytokines. The peptide was synthesized by the solid-phase method, and is identical to the natural peptide in amino acid composition, sequence and chemotactic potency. MDNCF forms two loops via a neighboring pair of disulfide bridges, the probable locations of which are residues 7-34 and 9-50. Reduction and alkylation eliminated chemotactic activity. MDNCF fragments 7-37, 30-72 and 17-72 were all biologically inactive. The data suggest that the region of the clustered pair of disulfide bridges is important for biological activity.

This study was undertaken to determine whether cell cooperation, either among monocytes or between monocytes and lymphocytes, is a prerequisite for monocyte chemotactic responsiveness. We compared Ficoll-Hypaque-separated mononuclear cells and a preparation of 99% pure monocytes obtained by chemotaxis in a newly designed separation chamber. Monocytes of both preparations migrated to chemoattractants without a lag phase, and no further increase in migrated cells was observed after 70 min. The cell dose-response was linear for both preparations over a wide range of cell concentrations in the cell input well of the chemotaxis chamber, suggesting that no monocyte-monocyte interaction was required. Since only 20 to 60% of the monocytes purified by chemotaxis migrated a second time, the possibility of a requirement for an accessory cell was tested. The addition to purified monocytes of several different mononuclear cell preparations comprising lymphocytes or nonmigrating monocytes had no effect on monocyte migration. These experiments show that normal human blood monocytes in vitro do not require stimuli from other cells to respond to chemoattractants. Their behavior is profoundly different from that of mouse peritoneal macrophages, which exhibit a time lag in vitro before migration toward an attractant and become more responsive with either increasing cell concentration or addition of purified lymphocytes.

Products of bacteria are potent chemoattractants for mammalian leukocytes. Several reports suggest that these attractants are small peptides. We compared the properties of culture fluids of Staphylococcus aureus with fMet-Leu-Phe, considered a prototype of bacterial attractant. Chemotactic activity for human monocytes of Staph. aureus culture filtrates was determined in multiwell chemotaxis chambers. At optimal concentrations, the filtrate attracted almost twice as many monocytes as fMet-Leu-Phe (53 +/- 5% of input number compared with 30 +/- 3%, in a series of ten experiments). Gel-filtration characteristics and susceptibility to proteolytic digestion suggested that chemotactic activity was due to peptides with a molecular size range of 500-2,000 daltons. Reverse-phase high-pressure liquid chromatography (HPLC) of unfractionated filtrate revealed nine peaks of chemotactic activity, most of which was in five of the peaks. One peak accounted for 40% of total activity. Individual peaks, like the unfractionated material, were capable of attracting about twice as many monocytes as the optimal concentration of fMet-Leu-Phe. Quantitative bioassay of the HPLC peaks showed that only 5% of the total Staph. aureus chemotactic activity eluted in the position of fMet-Leu-Phe. This is in contrast to the report that fMet-Leu-Phe accounted for 70% of neutrophil lysosomal enzyme-releasing activity of Escherichia coli culture fluid. In summary, chemotactic activity for monocytes of Staph. aureus culture fluid is due to peptides other than fMet-Leu-Phe; these peptides recruit a higher percentage of monocytes than fMet-Leu-Phe.

We designed a 48-well chemotaxis chamber to minimize manipulation time and amount of material required by the larger blindwell or Boyden chemotaxis chamber. Cell and chemoattractant dose-response curves showed that results were comparable to our better than those obtained with blindwell chambers. The volume of chemoattractant per well is 25 microliter; the number of cells can be as low as 10,000. The time needed for setting up this multiwell unit and for staining the membrane filter sheet is negligible. Combined with the use of an image analyzer to count the number of migrated cells, the method is suitable for clinical research on the functional state of monocytes in large groups of patients.

The cDNA coding for human monocyte-derived neutrophil-specific chemotactic factor (MDNCF) was cloned from LPS-stimulated human monocyte mRNA. The cDNA sequence codes for a polypeptide consisting of 99 amino acids, including a putative signal sequence. Comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of natural MDNCF shows that the mature functional protein comprises 72 amino acids, beginning with serine at residue 28. The deduced amino acid sequence shows striking similarity to several platelet-derived factors, a v-src-induced protein, a growth-regulated gene product (gro), and an IFN-gamma inducible protein. The availability of the MDNCF cDNA enabled us to use it as a probe to identify inducers of MDNCF mRNA expression in human PBMC. MDNCF mRNA was increased greater than 10-fold within 1 h after stimulation with LPS, IL-1, or TNF, but not by IFN-gamma, IFN-alpha, or IL-2. Furthermore, we also determined that LPS, IL-1, and TNF stimulated the mononuclear cells to produce biologically active MDNCF. This observation may account for the in vivo capacity of IL-1 and TNF to induce netrophil infiltrates.