Effect of lung surfactant collectins on bronchoalveolar macrophage interaction with Blastomyces dermatitidis: inhibition of tumor necrosis factor alpha production by surfactant protein D

Alveolar surfactant modulates the antimicrobial function of bronchoalveolar macrophages (BAM). Little is known about the effect of surfactant-associated proteins in bronchoalveolar lavage fluid (BALF) on the interaction of BAM and Blastomyces dermatitidis. We investigated BALF enhancement or inhibition of TNF-alpha production by BAM stimulated by B. dermatitidis. BAM from CD-1 mice were stimulated with B. dermatitidis without or with normal BALF, surfactant protein A-deficient (SP-A-/-) or surfactant protein D-deficient (SP-D-/-) BALF, or a mixture of SP-A-/- and SP-D-/- BALF. An enzyme-linked immunosorbent assay was used to measure tumor necrosis factor alpha (TNF-alpha) in culture supernatants. BALFs were standardized in protein concentration. BAM plus B. dermatitidis (BAM-B. dermatitidis) TNF-alpha production was inhibited > or = 47% by BALF or SP-A-/- BALF (at 290 or 580 microg of protein/ml, P < 0.05 to 0.01); in contrast, SP-D-/- BALF did not significantly inhibit TNF-alpha production. If SP-A-/- BALF was mixed in equal amounts with SP-D-/- BALF, TNF-alpha production by BAM-B. dermatitidis was inhibited (P < 0.01). Finally, pure SP-D added to SP-D-/- BALF inhibited TNF-alpha production by BAM-B. dermatitidis (P < 0.01). B. dermatitidis incubated with BALF and washed, plus BAM, stimulated 63% less production of TNF-alpha than did unwashed B. dermatitidis (P < 0.05). SP-D was detected by anti-SP-D antibody on BALF-treated unwashed B. dermatitidis in an immunofluorescence assay (IFA). The BALF depleted by a coating of B. dermatitidis lost the ability to inhibit TNF-alpha production (P < 0.05). 1,3-beta-Glucan was a good stimulator of BAM for TNF-alpha production and was detected on B. dermatitidis by IFA. beta-Glucan incubated with BALF inhibited the binding of SP-D in BALF to B. dermatitidis as demonstrated by IFA. Our data suggest that SP-D in BALF binds beta-glucan on B. dermatitidis, blocking BAM access to beta-glucan, thereby inhibiting TNF-alpha production. Thus, whereas BALF constituents commonly mediate antimicrobial activity, B. dermatitidis may utilize BALF constituents, such as SP-D, to blunt the host defensive reaction; this effect could reduce inflammation and tissue destruction but could also promote disease.     

Fungicidal mechanisms of activated macrophages: evidence for nonoxidative mechanisms for killing of Blastomyces dermatitidis.

The mechanism(s) by which lymphokine-activated peritoneal macrophages kill Blastomyces dermatitidis was studied. Resident peritoneal macrophages from BALB/cByJ mice, when treated overnight with lymph node cells plus concanavalin A, supernatants from concanavalin A-stimulated spleen cells, or recombinant gamma interferon, were then able to kill a virulent B. dermatitidis isolate (ATCC 26199) (at levels of 25% +/- 4%, 28% +/- 8%, and 21% +/- 5%, respectively). Killing was not significantly decreased or enhanced in the presence of superoxide dismutase (450 U/ml), catalase (20,000 U/ml), dimethyl sulfoxide (300 mM), or azide (1 mM). Viable B. dermatitidis elicited a brisk oxidative burst and superoxide anion production in activated macrophages as measured by lucigenin-enhanced chemiluminescence, e.g., 10(4) cpm. However, these responses were not significantly different from those of control macrophages. Luminol-enhanced chemiluminescence responses by activated or control macrophages were meager (less than or equal to 10(2) cpm). These results indicate that activated macrophages kill B. dermatitidis by a mechanism(s) independent of products of the oxidative burst.     

Recurrent miscarriage and variant alleles of mannose binding lectin, tumour necrosis factor and lymphotoxin alpha genes.

Variant alleles of the mannose binding lectin (MBL) gene are associated with increased susceptibility to infection and polymorphisms of tumour necrosis factor and lymphotoxin alpha genes (TNF, LTA) are associated with increased severity of infection. Studies have associated recurrent miscarriage with low serum mannose binding lectin concentrations and premature membrane rupture and preterm delivery with elevated maternal and fetal levels of TNF and the TNF (- 308) polymorphism. In this study the frequencies of variant MBL, TNF and LTA alleles in 76 Caucasian couples with idiopathic recurrent miscarriage were compared with those in 69 Caucasian control couples with no history of miscarriage and at least one previous live birth. A new assay based on hybridization to immobilized sequence-specific oligonucleotides (SSO) was used to rapidly detect nine MBL, two TNF and two LTA sequence variants. The assay genotyped all the structural and promoter MBL variants known to influence serum MBL concentrations. This assay was more reliable than restriction digestion or nested allele-specific PCR for the structural variants at codon 54 or 52, respectively. Reliability for codon 57 alleles was not assessed because of the low frequency in this population. The MBL haplotype frequencies in antenatal controls were similar to those reported in other control populations. The frequencies of structural variant MBL genes and of low, medium and high MBL level haplotypes were similar in the recurrent miscarriage and control couples. The TNF and LTA haplotype frequencies were similar in the recurrent miscarriage and control couples. In this carefully defined population no association has been found between recurrent miscarriage and variant alleles of the MBL, TNF or LTA genes.     

Orientia tsutsugamushi inhibits tumor necrosis factor alpha production by inducing interleukin 10 secretion in murine macrophages

Orientia tsutsugamushi (OT) is the causative agent of scrub typhus or Tsutsugamushi disease. We have previously reported that OT suppresses the production of inflammatory cytokines in murine macrophages. In this study, we examined the mechanism of OT to suppress the tumor necrosis factor alpha (TNF-alpha) production. J774 macrophages were preinfected with OT for various times and then treated with lipopolysaccharide (LPS) for 24 h. Preinfection by OT inhibited LPS-induced production of TNF-alpha, but did not affect the activation of NF-kappaB. This suppression was also induced by the conditioned medium (CM) from OT-infected macrophages. Interestingly, the CM contained a potent interleukin-10 (IL-10)-inducing factor that is active on activated macrophages. Therefore, the IL-10-inducing factor might enhance the negative-feedback mechanism ascribed to IL-10, to allow bacterial survival in the hostile environment of macrophages.     

Inhibition by delta-9-tetrahydrocannabinol of tumor necrosis factor alpha production by mouse and human macrophages.

Suppression by delta-9-tetrahydrocannabinol (THC) of tumor necrosis factor (TNF) production by macrophages has not been reported previously. The present study evaluated the effect in vitro of THC on soluble TNF-alpha production by cultured murine peritoneal macrophages. THC at 5 or 10 micrograms/ml added to medium [RPMI 1640 containing 10 ng LPS/ml, mouse IFN-gamma (100 u/ml), and 0.5% bovine serum albumin (BSA)] used to induce TNF significantly decreased TNF-alpha production by BALB/c mouse macrophages. Macrophages pretreated with THC at 0.1, 0.5, or 1.0 micrograms/ml in protein-free medium for 3 h at 37 degrees C, prior to TNF induction, also showed a decreased ability to produce TNF-alpha in a dose-dependent manner. Increasing the protein concentration from 0.5 to 5% BSA in the medium which was used to induce TNF prevented the inhibitory activity of THC. Human peripheral blood adherent cells treated with THC-containing medium produced less TNF-alpha than controls that were not exposed to THC. Thus, our data provide evidence that THC can inhibit TNF production by mouse and human macrophages. The drug's activity is concentration dependent and is related to the amount of serum protein in the medium used to induce this cytokine.     

Human macrophages acquire a hyporesponsive state of tumor necrosis factor alpha production in response to successive Mycobacterium avium serovar 4 stimulation.

Human macrophages (M phi) from most donors respond to inoculation with Mycobacterium avium serovar 4 (M. avium) by tumor necrosis factor alpha (TNF-alpha) production, which is of critical importance for proper defense against microorganisms. An initial infection of M phi with M. avium results in an incapacity to accumulate TNF-alpha mRNA after reinfection with M. avium, indicating adaptation to a hyporesponsive state by preexposure of the cells to M. avium. Adaptation to stimulation with M. avium is abrogated by the cyclooxygenase inhibitor indomethacin. In the presence of prostaglandin E2, indomethacin-exposed, M. avium-treated M phi remain unresponsive to a subsequent M. avium stimulus to increase steady-state TNF-alpha mRNA, suggesting that prostaglandin E2 is instrumental for the adaptation to an M. avium challenge. TNF-alpha mRNA accumulation induced by a second M. avium stimulus in the presence of indomethacin is blocked by the protein tyrosine kinase inhibitor herbimycin. In contrast, the initial M phi response to M. avium is inhibited by staurosporin, an inhibitor of phospholipid Ca(2+)-dependent protein kinases, indicating that the initial and the successive TNF-alpha responses to M. avium are dependent on different mechanisms.     

Role of MyD88 in diminished tumor necrosis factor alpha production by newborn mononuclear cells in response to lipopolysaccharide

Human newborns are more susceptible than adults to infection by gram-negative bacteria. We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS). In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-alpha) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation. At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-alpha. However, in the absence of plasma, MNC from newborns secreted significantly less TNF-alpha compared to MNC from adults. Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-alpha secretion was significantly lower for newborn MNC compared to adult MNC. Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry. However, the intensity of the CD14 marker was greater for adult than for newborn cells. Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells. The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IkappaBalpha phosphorylation, as well as IkappaBalpha degradation. Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88). We found a direct relation between adult and newborn TNF-alpha secretion and MyD88, which was significantly decreased in newborn monocytes. Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-alpha secretion in newborn monocytes.     

In vivo and in vitro cell-mediated immune responses to a cell wall antigen of Blastomyces dermatitidis.

An alkali-soluble, water-soluble cell wall fraction of Blastomyces dermatitidis, designated B-ASWS, was evaluated as an antigen for detecting in vivo (skin tests) and in vitro migration inhibition factor (MIF) production and lymphocyte transformation (LT) responses in Blastomyces-infected guinea pigs. The biological activity of B-ASWS was compared with that of blastomycin KCB-26. The superiority of B-ASWS, in terms of its sensitivity and specificity, was evident in in vivo and in vitro assays. Skin tests responses were obtained in 21 of the 24 Blastomyces-infected guinea pigs, whereas only one of the 14 Histoplasma-infected guinea pigs were significantly greater than those obtained using cell populations from Histoplasma-infected or noninfected guinea pigs. The con-MIF and LT in peritoneal exudate cells and lymph node cells of homologuosly infected animals. In each biological system, the response of the Blastomyces-infected guinea pigs were significantly greater than those obtained using cell populations from Histoplasma-infected or non-infected guinea pigs. The contrasting efficacy of B-ASWS as compared with blastomycin KCB-26, suggests that the cell wall antigen will be a useful tool for detecting cell-mediated immune responses in blastomycosis.     

Role of alveolar macrophages in innate immunity in neonates: evidence for selective lipopolysaccharide binding protein production by rat neonatal alveolar macrophages

As the first line of defense against inhaled substances, alveolar macrophages (AM) play a crucial role in maintaining lung homeostasis. This is achieved via phagocytosis of foreign material and the secretion of a wide range of mediator molecules, including those involved in neutrophil recruitment. Neonates are known to manifest increased susceptibility to lung infections, and we hypothesize that this may be due in part to a deficiency in the function of AM. We report here that although recruitment of neutrophils into the respiratory tract of newborn animals in response to Moraxalla catarrhalis exposure is greatly delayed and diminished, AM from newborn animals have greater phagocytic capacity when compared with those from adult animals. Additionally, newborn AM respond normally to lipopolysaccharide (LPS) via production of a variety of chemokines, including macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, monocyte chemotactic protein-1, gro/ cytokine-induced neutrophil chemoattractant, MIP-2, and tumor necrosis factor-alpha. We have also demonstrated an LPS inducible expression of messenger RNA for LPS binding protein (LBP) in neonatal AM that was not observed in AM from adult animals or in peritoneal macrophages. We speculate that local production of LBP by AM may be a significant factor in the neonatal immunologic response to infections, providing a compensatory mechanism for the deficiency in specific neonatal immunity during this period of development when the newborn is being exposed to a range of potentially pathogenic materials for the first time.     

Modulation of tumor necrosis factor production by macrophages in Entamoeba histolytica infection.

The macrophage-derived mediator tumor necrosis factor alpha (TNF) is a cytokine with pleiotropic effects. TNF exhibits potent immunologic and inflammatory properties in parasitic diseases. The present study examined the production of TNF by macrophages isolated from gerbils infected with Entamoeba histolytica and by naive macrophages in response to amoebae in vitro. Amoebic liver abscess-derived macrophages produced low constitutive basal levels of TNF; in response to lipopolysaccharide (LPS) stimulation, TNF production was enhanced by 14-, 11-, and 6-fold at 10, 20, and 30 days postinfection, respectively. Amoebic liver abscess-derived macrophages pretreated with either recombinant gamma interferon (IFN-gamma) or the cyclooxygenase inhibitor indomethacin augmented TNF production in response to soluble amoebic proteins and LPS. Kupffer cells and peritoneal and spleen macrophages from infected animals did not release TNF constitutively in vitro. However, TNF production in response to LPS stimulation was significantly higher at 10 and 20 days postinfection. Macrophages from infected and naive animals pretreated with recombinant IFN-gamma or indomethacin produced increased amounts of TNF in response to LPS but not in response to soluble amoebic protein stimulation. Pretreatment of naive macrophages with amoebic proteins inhibited LPS-induced TNF production by 69 to 79%; the effect of the amoebic proteins was partially reversed by indomethacin pretreatment. In contrast, IFN-gamma- and LPS-activated naive macrophages produced enhanced levels of TNF in response to live amoebae and soluble amoebic proteins. Our results demonstrate that TNF production by macrophages is altered during E. histolytica infection and in response to amoebae and suggest a role for IFN-gamma and prostaglandin E2 in regulating TNF production during the infection.     

Production of tumor necrosis factor alpha by resident and activated murine macrophages.

Alveolar macrophages (Am phi s), resident peritoneal macrophages (RPm phi s), and thioglycolate-elicited peritoneal macrophages (TGPm phi s) were isolated from C57BL/6 mice and incubated with lipopolysaccharide (LPS), stimulated cell supernatant, or recombinant interferon gamma (IFN-gamma) for 24 h. Tumor necrosis factor (TNF) in cell-free supernatants was measured by enzyme-linked immunosorbent assay. Amo phi s incubated with 10(3) ng/ml LPS produced 50 times more TNF than RPm phi s and 5 times more than TGPm phi s, and LPS alone induced maximum TNF production by Am phi s. Stimulated cell supernatant or recombinant IFN-gamma alone did not induce TNF production. A combination of LPS with stimulated cell supernatant or IFN-gamma had only a limited synergistic effect on TNF production by Am phi s. However, both LPS and stimulated cell supernatant or recombinant IFN-gamma induced maximum TNF production by RPm phi s and TGPm phi s. TGPm phi s showed greater sensitivity to LPS and stimulated cell supernatant or IFN-gamma with regard to TNF production than the other macrophage populations investigated.     

Mycolactone-mediated inhibition of tumor necrosis factor production by macrophages infected with Mycobacterium ulcerans has implications for the control of infection

The pathogenicity of Mycobacterium ulcerans, the agent of Buruli ulcer, depends on the cytotoxic exotoxin mycolactone. Little is known about the immune response to this pathogen. Following the demonstration of an intracellular growth phase in the life cycle of M. ulcerans, we investigated the production of tumor necrosis factor (TNF) induced by intramacrophage bacilli of diverse toxigenesis/virulence, as well as the biological relevance of TNF during M. ulcerans experimental infections. Our data show that murine bone marrow-derived macrophages infected with mycolactone-negative strains of M. ulcerans (nonvirulent) produce high amounts of TNF, while macrophages infected with mycolactone-positive strains of intermediate or high virulence produce intermediate or low amounts of TNF, respectively. These results are in accordance with the finding that TNF receptor P55-deficient (TNF-P55 KO) mice are not more susceptible than wild-type mice to infection by the highly virulent strains but are more susceptible to nonvirulent and intermediately virulent strains, demonstrating that TNF is required to control the proliferation of these strains in animals experimentally infected by M. ulcerans. We also show that mycolactone produced by intramacrophage M. ulcerans bacilli inhibits, in a dose-dependent manner, but does not abrogate, the production of macrophage inflammatory protein 2, which is consistent with the persistent inflammatory responses observed in experimentally infected mice.     

Preexposure of murine macrophages to CpG oligonucleotide results in a biphasic tumor necrosis factor alpha response to subsequent lipopolysaccharide challenge

Bacterial DNA and synthetic oligonucleotides containing CpG sequences (CpG-DNA and CpG-ODN) provoke a proinflammatory cytokine response (tumor necrosis factor alpha [TNF-alpha], interleukin-12 [IL-12], and IL-6) and increased mortality in lipopolysaccharide (LPS)-challenged mice via a TNF-alpha-mediated mechanism. It was hypothesized that preexposure of macrophages to CpG-ODN would result in an increased TNF-alpha response to subsequent LPS challenge in vitro. Using the murine macrophage cell line RAW 264.7, we demonstrated both a rapid proinflammatory cytokine response (TNF-alpha) and a delayed inhibitory cytokine response (IL-10) with CpG-ODN. Preexposure of macrophages to CpG-ODN for brief periods (1 to 3 h) augmented TNF-alpha secretion and mRNA accumulation following subsequent LPS challenge (1 microg/ml). However, prolonged preexposure to CpG-ODN (6 to 9 h) resulted in suppression of the TNF-alpha protein and mRNA response to LPS. The addition of anti-IL-10 antibody to CpG-ODN during preexposure resulted in an increase in the LPS-induced TNF-alpha response over that induced by CpG-ODN preexposure alone. Thus, while brief preexposure of macrophages to CpG-ODN augments the proinflammatory cytokine response to subsequent LPS challenge, prolonged preexposure elicits IL-10 production, which inhibits the TNF-alpha response. Although the initial proinflammatory effects of CpG-DNA are well established, the immune response to CpG-DNA may also include autocrine or paracrine feedback mechanisms, leading to a complex interaction of proinflammatory and inhibitory cytokines.     

Augmentation of the neutrophil response to Naegleria fowleri by tumor necrosis factor alpha.

Conditioned medium from phytohemagglutinin-stimulated human mononuclear leukocytes, previously shown to activate neutrophils for amoeba killing, was found to contain high levels of tumor necrosis factor alpha (TNF-alpha) by an enzyme-linked immunosorbent assay. The effects of human recombinant TNF-alpha on the response of human neutrophils to the pathogenic free-living amoeba Naegleria fowleri was studied in vitro. The data showed that recombinant human TNF-alpha augmented the neutrophil respiratory burst (assessed by the cytochrome c reduction assay and lucigenin-dependent chemiluminescence assay) in response to amoebae opsonized with human serum. The priming effects of TNF-alpha were transient; marked enhancement was found with short 5- to 30-min preincubations of neutrophils with the cytokine. The enhancement of oxygen radical production was evident with 20 U of TNF-alpha per 10(6) neutrophils and continued to increase with up to 100 U. TNF-alpha also augmented the neutrophil lysosomal enzyme release in response to N. fowleri. The results support previous reports suggesting an important role of neutrophil cytokine activation for effective immunity against free-living amoebae.     

The effect of canine macrophages on the adherence and growth of Blastomyces dermatitidis yeast: evidence of a soluble factor that enhances the growth of B. dermatitidis yeast

Blastomycosis is a medically important systemic fungal infection of dogs and humans. Phagocytic cells are the first line of cellular defence against B. dermatitidis, and are a prominent feature in the lesions and exudate of canine blastomycosis. The adherence of B. dermatitidis yeast to canine phagocytes, and the effects of such adherence on the growth of B. dermatitidis yeast, has not been previously reported. The results of this study demonstrate that canine complement enhances the adherence of B. dermatitidis yeast to canine macrophages. Initiation of the canine complement cascade by B. dermatitidis yeast appeared to occur predominantly by the classical pathway. Adherence of B. dermatitidis yeast to canine macrophages enhanced the growth of the yeast. In the absence of macrophages, this effect could be duplicated by incubating yeast in conditioned medium from co-cultures of macrophages and yeast. This observation suggests that a soluble factor is involved in the growth enhancement of the yeast, These findings provide new insights into the adherence of B. dermatitidis yeast to canine macrophages, and how adherence influences the proliferation of B. dermatitidis yeast.     

Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology

Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused by Mycobacterium tuberculosis. The mechanisms involved in the containment of latent tuberculosis are poorly understood. Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-alpha), we examined the effects of TNF-alpha on the immunological response of the host in both persistent and reactivated tuberculous infections. The results confirm an essential role for TNF-alpha in the containment of persistent tuberculosis. TNF-alpha neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space. Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed. Interleukin-12p40 and gamma interferon gene expression in TNF-alpha-neutralized mice was similar to that in control mice. In contrast, interleukin-10 expression was augmented in the TNF-alpha-neutralized mice. In summary, results of this study suggest that TNF-alpha plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.     

Neutralization of tumor necrosis factor alpha suppresses antigen-specific type 1 cytokine responses and reverses the inhibition of mycobacterial survival in cocultures of immune guinea pig T lymphocytes and infected macrophages

Neutralization of tumor necrosis factor alpha (TNF-alpha) significantly down-regulated antigen-induced lymphoproliferation and the expression of interleukin-12 p40 and gamma interferon mRNA and enhanced the viability of intracellular attenuated and virulent mycobacteria in cocultures of immune T cells and macrophages obtained from Mycobacterium bovis BCG-vaccinated guinea pigs. This suggests the crucial role of TNF-alpha in the activation of a type 1 T-cell response against Mycobacterium tuberculosis infection.     

Blastomyces dermatitidis chemotactic factor: kinetics of production and biological characterization evaluated by a modified neutrophil chemotaxis assay

Chemotactic activity for human polymorphonuclear neutrophils (PMNs) was detectable in culture filtrates (CFs) of Blastomyces dermatitidis and may have influenced the pathogenesis of blastomycosis. Production of this chemotaxin depended upon culture age and medium; peak levels were achieved after incubation for 17 days or more in minimal essential medium. This factor was also chemotactic for human monocytes. CF was temperature stable even after treatment at 100 degrees C for 60 min. The activity was stable under alkaline conditions but was destroyed below pH 7. Dialyzed, chemotactically active CF contained approximately 60 micrograms of carbohydrate per ml; total protein was estimated to be less than 0.8 micrograms/ml. Preincubation of PMNs with CF or N-formyl-L-methionyl-L-leucyl-L-phenylalanine deactivated their chemotactic response to each agent, whereas the chemotactic response to zymosan-activated serum was not affected. In addition, deactivation with N-formyl-L-methionyl-L-leucyl-L-phenylalanine reduced the response to CF. Pretreatment of CF with PMNs decreased chemotactic activity, which may reflect binding of the chemotaxin molecules to PMN receptors. A modified chemotaxis assay was developed in which commercial, disposable multiwell plates are used. This method was rapid, efficient, and inexpensive and permitted the assay of larger numbers of samples than was previously feasible with conventional chemotaxis methods.