Inhibition of leukocyte chemotaxis by serum factor in diabetes mellitus: Selective depression of cell responses mediated by complement-derived chemoattractants

Rat neutrophil chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene (LT) B₄, and lipopolysaccharide-activated serum (LPS-AS) were quantitatively assessed using the micropore filter system. Cells were suspended in either normal or diabetic rat serum for testing. Diabetic donor serum did not affect migration of neutrophils in a concentration gradient of the synthetic chemotactic agents. In contrast, the migratory responses to LPS-AS were significantly less than normal in this circumstance. Summation of effects was observed when FMLP and LPS-AS, or LTB₄ and LPS-AS were simultaneously added to the test chamber, with cells suspended in normal serum. Suspended in diabetic rat serum neutrophils responded normally to the synthetic chemoattractants but the response to the activated serum was blocked. Cells previously incubated in the presence of diabetic donor serum then transferred to a culture medium for testing, presented reduced migratory responses to LPS-AS. Supramaximal, inhibitory concentrations of FMLP and LTB₄, did not influence the response of neutrophils to LPS-AS.In vivo, suppression of cellular emigration to an inflamed area was observed from the early stages of the diabetic state. The inhibitory activity of chemotaxis in diabetes mellitus was previously reported to be associated with a protein factor in plasma of the animals. It is suggested that the inhibitory factor of chemotaxis in diabetes mellitus interacts with neutrophil receptors for complement-derived chemoattractants to induce blockade of cell-oriented locomotion eitherin vitro orin vivo.     

Chemotactic factor-induced effects upon deformability of human polymorphonuclear leukocytes

We have compared the effects of the synthetic chemotactic peptide,N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe), on the deformability and chemotaxis of human polymorphonuclear leukocytes (PMNs). By utilizing the technique of cell elastimetry (deformability), which measures the negative pressure required to aspirate cells into a micropipet, we have shown a correlation between f-Met-Leu-Phe concentrations and PMN deformability which parallels the effects of that chemoattractant on filter chemotaxis. At concentrations as low as 10^–11 M, a change in the deformability characteristics of a population of neutrophils was demonstrated. The increase in deformability became most marked at peptide concentrations in the range of 1–5×10^–9 M, corresponding to the optimal concentration range in the filter chemotaxis assay. Concentrations of 10^–8 M and greater were associated with a significant increase in the negative pressure required to aspirate the cells into a micropipet. Alterations in PMN deformability, therefore, occur as a natural consequence of stimulation of a human PMN with a chemotactic factor and may be a prerequisite to efficient movement.     

Modulation of human neutrophil and monocyte chemotaxis and superoxide responses by recombinant TNF-alpha and GM-CSF

The effects of recombinant TNF and GM-CSF on human peripheral blood neutrophil and monocyte chemotaxis and the superoxide response were studied. TNF exhibited a slight chemotactic activity for both cell types. Preincubation of neutrophils with as little as 40 units/ml strongly inhibited the neutrophil chemotaxis towards f-Met-Leu-Phe. The inhibition of monocyte chemotaxis required higher concentrations of TNF (greater than 400 units/ml). TNF at concentrations higher than 500 units/ml enhanced the generation of superoxide anions by neutrophils stimulated with f-Met-Leu-Phe. In contrast, TNF even at 2,000 units/ml did not prime monocytes for enhanced superoxide response. GM-CSF alone did not exhibit any chemotactic activity for any of the cell types tested. Preincubation of cells with GM-CSF inhibited chemotaxis of neutrophils but not of monocytes. GM-CSF was as potent as TNF in enhancing the generation of superoxide response by neutrophils. However, GM-CSF did not have any effect on monocyte superoxide response. The priming ability of TNF and GM-CSF on neutrophils was heat-sensitive. We conclude that TNF and GM-CSF play a more pronounced regulatory role on neutrophils than on monocytes. Inhibition of neutrophil chemotaxis followed by enhancement of the superoxide response by TNF and GM-CSF may provide an attractive mechanism by which these cytokines assist in fighting invading microorganisms.     

Inhibition of human neutrophil receptor-mediated uptake of N-formyl-met-leu-phe by platelet factor 4 (59-70).

Human platelet factor 4 (PF4) and a substituent dodecapeptide designated PF4(59-70) elicited human neutrophil and monocyte chemotaxis with a similar concentration-dependence and maximal responses equal to that attained by chemotactic fragments of C5 (C5fr). At maximally chemotactic concentrations, PF4(59-70) stimulated the secretion by neutrophils of approximately 40% and 60% of the respective quantities of beta-glucuronidase and beta-glucosaminidase released by 10(-6) M N-formyl-methionyl-leucyl-phenylalanine (fMLP). In contrast to the deactivation of chemotaxis achieved by preincubation of neutrophils with other chemotactic factors, prior exposure to 10(-6)M PF4(59-70) for 2 min, or 20 min at 37 degrees, enhanced by 1.5- to 2-fold the chemotactic responses of neutrophils evoked by optimal concentrations of fMLP, C5fr, leukotriene B4, and PF4(59-70). Concentrations of PF4(59-70) which enhanced neutrophil chemotaxis inhibited the rate of receptor-mediated internalization of [3H]fMLP at 37 degrees and 18 degrees, but at 0 degrees failed to alter the binding affinity or the number of receptors for [3H]fMLP. Preincubation of neutrophils at 37 degrees with concentrations of PF4(59-70) which enhanced neutrophil chemotaxis also did not affect the subsequent binding of [3H]fMLP at 0 degrees. The inhibition by PF4(59-70) of the receptor-mediated internalization of [3H]fMLP was not mimicked by other positively charged compounds. The specific inhibition of receptor-mediated internalization of fMLP may explain the enhanced chemotactic responsiveness of neutrophils preincubated with PF4(59-70).     

A synthetic peptide which specifically inhibits heat-treated interleukin-8 binding and chemotaxis for neutrophils

Interleukin-8 (IL-8) is a peptide which is secreted by stimulated human monocytes and which is chemotactic for human neutrophils. We synthesized three overlapping peptides spanning the amino-terminal region of the IL-8 sequence. None of the peptides retained the chemotactic activity of the native molecule. One of the peptides, IL-8(_3–25), inhibited the neutrophil chemotactic activity of recombinant IL-8 (rIL-8) which had been preheated to 40°C but did not reduce neutrophil chemokinesis, or the chemotactic activity of unheated rIL-8, FMLP, C5a or LTB₄. Interleukin-8 exhibited similar binding kinetics and chemotaxis for neutrophils regardless of whether it had been pretreated at 40°C.In addition, IL-8(_3–25) was also able to decrease the binding of prehead IL-8 to neutrophils. IL-8(_3–25), which can self-associate, binds directly to receptors on the neutrophil. The data suggest that heat-treated, but not untreated, IL-8 causes the IL-8(_3–25) multimers to disaggregate, allowing the monomeric peptide to directly bind to the IL-8 receptor and thus inhibiting IL-8/receptor binding.     

Chemotactic factor-induced enhancement of the binding of human immunoglobulin classes and subclasses to neutrophils and eosinophils

Sheep erythrocytes (E) coated with either human IgG1, IgG2, IgG3 or IgG4 myeloma proteins formed rosettes with human neutrophils and eosinophils. Prior incubation with the chemotactic agents f-met-leu-phe or leukotriene B4 enhanced neutrophil and eosinophil E-IgG1 rosettes in a time- and dose-dependent fashion. There were comparable increases in neutrophil and eosinophil f-met-leu-phe-induced rosettes with either E-IgG1, E-IgG2, E-IgG3 or E-IgG4 whereas this agent did not appear to influence E-IgA1, E-IgD, E-IgM or E-IgE binding to these granulocytes. These studies indicate that human neutrophil and eosinophil IgG (Fc) receptors are enhanced by chemotactic agents in a similar way to that previously described for complement receptors.     

Priming of human neutrophil functions by tumor necrosis factor: Enhancement of superoxide anion generation,degranulation, and chemotaxis to chemoattractants C5a and f-Met-Leu-Phe

Recombinant human tumor necrosis factor- (rTNF) stimulated increased generation of Superoxide anion (O ₂ ^– ) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2–2200 units/ml) for 10 min enhanced the subsequent generation of O ₂ ^– in response to C5a and f-MetLeu-Phe(FMLP). Recombinant TNF did not enhance O ₂ ^– generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001–50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001–0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100–10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.     

Effect of disodium cromoglycate on neutrophil movement and intracellular calcium mobilization

Disodium cromoglycate (DSCG) is a widely used drug in the treatment of allergy and asthma. Although its mode of action is not completely understood, it appears to prevent activation and release of mediators from mast cells. Neutrophils may also play a prominent role in clinical asthma and in other diseases of the airways. We have therefore studied the effect of DSCG on the activation of neutrophils from healthy adults. DSCG in concentrations of 1, 10, and 50 μg/ml significantly inhibited chemotaxis to zymosan-activated serum or formyl-methionyl-leucyl-phenylalanine. When leukotriene B₄ was used as the chemoattractant, no inhibition was found. Incubation of the cells with the drug for 30 minutes elicited the most pronounced inhibition. Since calcium is a key factor in the activation of cells, we used the calcium-specific probe Quin-2 to examine free levels of this cation after chemotactic-factor stimulation. Treatment of neutrophils with DSCG significantly reduced intracellular free calcium levels induced by zymosan-activated serum but not leukotriene B₄. Thus, it appears that DSCG may function not only to stabilize mast cells in allergy and clinical asthma but also may interfere with neutrophil activation and movement into the airways.     

Pasteurella haemolytica Leukotoxin-Induced Increase in Phospholipase A2 Activity in Bovine Neutrophils

Exposure of bovine neutrophils to Pasteurella haemolytica leukotoxin (LKT) stimulates the production of leukotriene B₄ (LTB₄), which is believed to be an important chemotactic agent in the development of acute fibrinopurulent pneumonic infection in cattle. The involvement of phospholipase A₂ (PLA₂) in LKT-induced synthesis of LTB₄ was studied by using bovine neutrophils labeled with ³H-arachidonate ([³H]AA). Incubation of isolated neutrophils with [³H]AA resulted in incorporation of radioactivity in the PLA₂ substrates phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Exposure of radiolabeled neutrophils to LKT caused concentration- and time-dependent release of radioactivity and redistribution of radioactivity in neutrophil membranes consistent with utilization of phosphoglyceride substrate and release of free fatty acid and eicosanoid products. These LKT-induced effects could be inhibited by pretreatment with arachidonyl trifluoromethyl ketone, an inhibitor of type IV cytoplasmic PLA₂, and were dependent on extracellular calcium. These results support the conclusion that LKT-induced synthesis of LTB₄ involves a calcium-mediated increase in PLA₂ activity.     

Relationship between chemical composition and biological function of Pseudomonas aeruginosa lipopolysaccharide: effect on human neutrophil chemotaxis and oxidative burst

There are conflicting data on the effect of bacterial lipopolysaccharides (LPS) on the function of human neutrophils. The present study was designed to examine the relationship between chemical composition and the modulatory effect of LPS on human neutrophil function. LPS was extracted from five strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients by the hot phenol-water method. Chemical characterization included neutral sugars, amino components, and fatty acids. Neutrophils isolated from peripheral blood of healthy individuals were preincubated with different concentrations of LPS. After preincubation, the chemotaxis and chemiluminescence of neutrophils to various stimuli were determined. It was shown that LPS from different strains did not exert the same degree of regulatory effect on neutrophil functions. LPS from strain 174-O:9 exerted the most pronounced effect on neutrophil function seen as inhibition of neutrophil chemotaxis toward the chemotactic peptide f-Met-Leu-Phe and zymosan-activated serum (ZAS) and priming of the cells for less than or equal to 8-fold enhancement of chemiluminescence response to f-Met-Leu-Phe. Conversely, LPS from strain 1118-O:3 had no effect on neutrophil chemotaxis and a slight effect on chemiluminescence. The major differences in chemical composition of the LPS from these two strains are in the rhamnose and heptose content of the O side chain and in the alanine content of the core region. These data indicate that chemical composition of the LPS molecule may play an important role in biological activity of LPS.     

Pharmacological profile of a novel,orally active leukotriene B4 antagonist,SM-15178

SM-15178, a new hydroxyacetophenone derivative, was evaluated to determine its antiinflammatory activity and antagonistic activity against leukotriene B₄ (LTB₄). SM-15178 inhibited [³H]LTB₄ binding to its receptors on human neutrophils (IC₅₀=0.30M). It inhibited LTB₄-induced chemotaxis of human neutrophils (IC₅₀ =0.72M) with little inhibitory effect against C5a or FMLP-induced chemotaxis at concentrations up to 30M. The compound alone did not cause human neutrophil chemotaxis at concentrations up to 10M. LTB₄-induced chemotaxis of mouse and rat neutrophils and guinea pig eosinophils was also inhibited by the compound, with IC₅₀ values of 0.55, 0.52, and 0.58 M, respectively. In an in vivo study, SM-15178, given orally, significantly prevented LTB₄-induced transient leukopenia. It also suppressed LTB₄-induced bronchoconstriction in the guinea pig almost completely when given orally at a dose of 40 mg/kg. Furthermore, orally given SM-15178 suppressed arachidonic acid-induced neutrophil infiltration in mouse ears and Arthus reaction-induced paw edema in the mouse in a dose-dependent manner. These results suggest that SM-15178 is a selective and orally active LTB₄ antagonist and that it might be effective for the treatment of some types of inflammatory diseases.     

Influence of extracellular Ca2+ and Mg2+ on chemotactic factor-induced neutrophil aggregation

The influences of extracellular Ca²⁺ and Mg²⁺ on chemotactic factor-induced rabbit polymorphonuclear neutrophil (PMN) aggregation was studied using a recently described Coulter counter assay technique. Compared with those of other PMN functional assays (adhesion, chemotaxis, degranulation, and phagocytosis), the cation requirements for cell aggregation appear unique. Chemotactic factor-induced aggregation did not occur in the absence of either cation. When the concentration of both cations was increased equivalently, aggregation increased. The effect plateaued at 2.8 mM of Ca²⁺ and Mg²⁺. When the concentration of one cation alone was increased, aggregation peaked. Further increases inhibited maximal aggregation. By systematic variation of the cation concentrations, optimal aggregation was found at Ca²⁺ and Mg²⁺ concentrations of 2.8 mM. At these concentrations, significant aggregation was induced with chemotactic doses of bacterial factor, the chemotactic fragment of human C5, and the synthetic chemotactic tripeptide, formyl-met-leu-phe. Thus, under these conditions, chemotactic factor-induced-aggregation of neutrophils may be a useful indicator of interactions of chemotactic factors with neutrophils.Supported in part by NIH Grants AI-13910 and HL-07202.Postdoctoral trainee supported by HL-05474.     

Preferential human eosinophil chemotactic activity of the platelet-activating factor (PAF) 1-0-hexadecyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC)

The chemotactic responses of human blood neutrophils and of eosinophils of two different densities, which were resolved by centrifugation on gradients of polyvinylpyrrolidone-coated silica gel (Percoll), were quantified in modified Boyden micropore filter chambers using highly purified synthetic 1-0-hexadecyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC or PAFacether) and leukotriene B₄ (LTB₄) as stimuli. Maximal chemotactic responses of the densest eosinophils, less dense eosinophils, and neutrophils were evoked by 1 nM, 100 nM, and 1 µM PAFacether, respectively, and by 30–100, 30–100, and 10 nM LTB₄. The magnitude of the maximal chemotactic response to PAFacether of the densest eosinophils was significantly greater than that of neutrophils. The eosinophil responses to PAFacether were chemotactic, as distinguished from chemokinetic, and were not influenced by the percentage of contaminating neutrophils. PAFacether is a more potent chemotactic factor for eosinophils than neutrophils and selectively attracts the densest population of human blood eosinophils.     

In Vivo Crevicular Leukocyte Response to a Chemotactic Challenge: Inhibition by Experimental Diabetes

Diabetes in rats inhibits the migration of neutrophils into the healing gingival crevice, an effect associated with impaired in vitro neutrophil chemotactic activity. We recently described the in vivo response of human and rat crevicular neutrophils to a chemotactic challenge and used this assay in the present study on streptozotocin-induced diabetic rats. Optimal concentrations of two chemotactic agents, casein (0.2 μl, 2 mg/ml) or N-formylmethionylleucylphenylalanine (0.2 μl, 10⁻⁴ M), were placed into the gingival crevices of control and diabetic rats (time zero) after the resting neutrophil count was measured. After a 15-min delay, the neutrophil counts and gingival crevicular fluid flow were assessed every 5 min for another 0.5 h. The control rats (n = 14) showed an increase in neutrophil counts which reached maximum levels 30 min after the N-formylmethionylleucylphenylalanine challenge (“peak” neutrophil response) and decreased dramatically 5 min later. Diabetes of 4 days (n = 4), 14 days (n = 8), and 20 days (n = 5) duration reduced the peak neutrophil response 45, 66, and 71%, respectively. Casein produced the same response as N-formylmethionylleucylphenylalanine in control rats. Uncontrolled diabetes of 20 days duration reduced the peak neutrophil response to casein by 83%; diabetics administered insulin on a daily basis showed a reduction of only 34%. The pattern of change in gingival crevicular fluid flow in response to chemoattractants paralleled the neutrophil response. The chemotactic activity of peritoneal neutrophils was assessed in vitro with the agarose gel technique and was found to be correlated (r = 0.84; P < 0.01) with the in vivo chemotactic response in the same rats. If the same in vivo defect is observed in humans with diabetes (or with other systemic diseases associated with leukocyte dysfunction), this test could be useful diagnostically to rapidly assess neutrophil chemotaxis in lieu of in vitro assays and to identify patients who are unusually susceptible to aggressive periodontal disease.     

Inhibition of neutrophil migration in mice by mouse formyl peptide receptors 1 and 2 dual agonist: indication of cross-desensitization in vivo

It has been reported that the stimulation of neutrophils with N‐formyl‐Met‐Leu‐Phe (fMLF), an agonist for formyl peptide receptor (Fpr) 1, renders cells unresponsive to other chemoattractants in vitro. This is known as cross‐desensitization, but its functional relevance in neutrophil migration in vivo has not been investigated. Here, we show that precedent stimulation of mouse neutrophils with compound 43, a non‐peptidyl agonist for mouse Fpr1 and Fpr2, rendered the cells unresponsive to a second stimulation with C5a, leukotriene B₄, or keratinocyte‐derived cytokine (KC) in calcium mobilization and chemotaxis assays in vitro. The expression of chemokine (C‐X‐C motif) receptor 2 (CXCR2) on the surface of neutrophils was concomitantly diminished by stimulating the cells with the compound. Moreover, oral administration of the compound to mice before they were exposed to lipopolysaccharide (LPS) aerosol resulted in a dose‐dependent reduction in the neutrophil count in bronchoalveolar lavage fluid. The expression of CXCR2 on blood neutrophils was also reduced in the compound‐administered mice. The recipient mice that underwent adoptive transfer of fluorescence‐labelled neutrophils that had been incubated with the compound showed a substantial decrease in neutrophil counts in bronchoalveolar lavage fluid after they were exposed to LPS, when compared with the control mice to which vehicle‐treated neutrophils had been transferred. These results are consistent with the idea that the agonist for Fpr1 and Fpr2 induced cross‐desensitization in neutrophils and attenuated neutrophil migration into the airways. Our results also revealed the unpredicted effect of an Fpr1 and Fpr2 dual agonist, which may act as a functional antagonist for multiple chemoattractant receptors in vivo.     

Neutrophil Migration in Opposing Chemoattractant Gradients Using Microfluidic Chemotaxis Devices

Neutrophils migrating in tissue respond to complex overlapping signals generated by a variety of chemotactic factors (CFs). Previous studies suggested a hierarchy between bacteria-derived CFs and host-derived CFs but could not differentiate neutrophil response to potentially equal host-derived CFs (IL-8 and LTB₄). This paper reports neutrophil migration in conflicting gradients of IL-8 and LTB₄ using a microfluidic chemotaxis device that can generate stable and well-defined gradients. We quantitatively characterized the movement of cells from time-lapse images. Neutrophils migrate more efficiently toward single IL-8 gradients than single LTB₄ gradients as measured by the effective chemotactic index (ECI). In opposing gradients of IL-8 and LTB₄, neutrophils show obvious chemotaxis toward a distant gradient, consistent with previous reports. When an opposing gradient of LTB₄ is present, neutrophils show less effective chemotaxis toward IL-8 than when they are in a gradient of IL-8 alone. In contrast, the chemotactic response of neutrophils to LTB₄ is not reduced in opposing gradients as compared to that in a single LTB₄ gradient. These results indicate that the presence of one host-derived CF modifies the response of neutrophils to a second CF suggesting a subtle hierarchy between them.This revised version was published online in May 2005 with corrected author affiliations     

A modified method for chemotaxis under agarose.

The chemotactic properties of homogeneus human C5a and C5a_{des Arg}, as well as other cytotaxins, can be evaluated accurately by using a modified ‘chemotaxis under agarose’ method that requires neither serum nor albumin. Gelatin, at a final concentration of 0.25%, substitutes adequately for the serum or albumin that has been used previously. By assessing both the directed and random migration of human neutrophils under a variety of conditions, we delineated characteristic features of the neutrophils' chemotactic response to 3 cytotaxins: C5a, C5a_{des Arg} and N-f-Met-Phe. Neutrophils responding to these stimuli move across the glass microscope slide that serves as a supporting surface in 3 distinctly different migratory patterns designated here as ‘rocket-shaped, blunt and desensitized’. These readily identifiable patterns each correlate with a unique neutrophil morphology and depend both on the nature and the concentration of the chemotactic factor present. From the distance the neutrophils migrate after various time intervals, we can calculate the mean initial rate of migration for the most rapidly moving cells. These studies show that human neutrophils migrate at an average initial rate of 5.0 ± 0.2 μ/min in response to each of the factors tested and at an average random rate of 1.7 ± 0.1 μ/min in the absence of any chemotactic stimulus. Optimum chemotaxis, as judged by ‘chemotactic index’ values, occurs when a total of 2.5 × 10⁵ neutrophils/10 μl is incubated for 2–2.5 h at 37°C. This modified assay is sufficiently sensitive for use in assessing the chemotactic properties of a wide variety of factors. In particular, it may become the preferred method for studying purified human C5a and C5a_{des Arg} because the latter factor fails to stimulate neutrophil migration in the Boyden chamber assay system in the absence of serum or serum proteins.     

Tolfenamic acid inhibits leukotriene B4-induced chemotaxis of polymorphonuclear leukocytes in vitro

Inhibition of prostanoid synthesis is usually regarded as the mode of action of nonsteroidal antiinflammatory drugs (NSAIDs). In addition, some NSAIDs have been reported to have prostanoid-independent inhibitory effects on neutrophil functions. In the present study, we examined the effects of acetylsalicylic acid, diclofenac, indomethacin, ketoprofen, piroxicam and tolfenamic acid on leukotriene B₄ (LTB₄)-induced chemotaxis of human polymorphonuclear leukocytes (PMNs) in vitro. Tolfenamic acid inhibited LTB₄-induced chemotaxis (IC₅₀ 59M), whereas the other compounds were ineffective. Tolfenamic acid inhibited also FMLP-induced chemotaxis at the same concentration range (IC₅₀ 46M). About 25% reduction in the chemotactic response was achieved with therapeutic concentrations of tolfenamic acid. We suggest that the inhibition of PMN chemotaxis is an additional mechanism in the antiinflammatory action of tolfenamic acid and that this action is not ligand specific.     

Regulation of neutrophil functions by elastase-generated IgG fragments.

IgG is split by neutrophil elastase into Fc and Fab fragments. These IgG fragments influence the functions of stimulated neutrophils such as chemotaxis, oxidative burst, and enzyme release. FMLP stimulated leukocyte chemotaxis is specifically inhibited by the elastase generated Fc fragments. Seven nmol Fc/10(6) PMN totally inhibit the chemotaxis stimulated by 16 to 125 nM FMLP. Native IgG and Fab fragments show no effect. FMLP-stimulated superoxide anion generation is specifically inhibited by Fc fragments with half maximal inhibition by 1.2 nmol/10(6) PMN. The generation of hydrogen peroxide is concomitantly stimulated, resulting in a superoxide dismutase-like effect. FMLP-stimulated elastase and myeloperoxidase release are enhanced by Fab fragments (10 nmol/10(6) PMN) to 206 and 155%, respectively, of reference values by 25 nM FMLP, while Fc and native IgG stimulate to a less extent. Consequently, elastase-generated Fc fragments have an inhibitory effect on inflammation by reducing chemotaxis and oxidative burst of stimulated neutrophils. The release stimulating activity of Fab fragments results in an up-regulation of elastase induced IgG degradation.     

Alloantibody bipolar bridging; A new mechanism of cell surface activation

Bipolar bridging of cellular membrane receptors and epitopes by alloantibodies (Fab bridging the MHC antigens and Fc the Fc receptors) has been shown on a murine mast cell model to be a way of cell signaling and activation. In order to test a possible general significance of this phenomenon, another model was studied, namely guinea pig neutrophils. It was found: (1) that neutrophils from S2, S13 and BIO-AD strains both express class I (B) and class II (Ia) antigens on their surface, as detected by a Prot.A-SRBC rosetting method, after cell incubation with related alloantibodies; (2) that Fc receptors for IgG (FcγR) were specific for IgG₂ subclass, as determined by the same rosetting method after binding of preformed immune complexes (IgG₁, IgG₂ and F(ab′)₂ anti-DNP-DNP₂₅ BSA); and (3) that specific alloantibodies of IgG₂ subclass were able to specifically activate the neutrophil oxidative metabolism as shown by superoxide anion (O₂^?) release, detected by the luminol-dependent chemiluminescence method. Neither the IgG₁ nor F(ab′)₂ portion were able to trigger O₂^? release.This demonstrates a second situation of a cell membrane activation through alloantibody bipolar bridging.