Dysfunctional LAD-1 neutrophils and colitis.

Leukocyte adhesion deficiency 1 (LAD-1) is characterized by absent or dysfunctional beta2 integrin (CD18), leading to defective chemotaxis, adherence, phagocytosis, and bacterial killing. Colitis, except for rare intestinal necrotizing events, is not a well-recognized feature of this immunodeficiency. A case of nonspecific colitis clinically resembling Crohn's disease in a patient with the severe form of LAD-1 (0.5% < CD18) has been previously reported. We describe an adult patient with the moderate form of LAD-1 and chronic colitis characterized by extensive inflammation and ulceration of the right colon and terminal ileum, leading to adhesions and strictures. The chronic colitis described in this article associated with the dysfunctional neutrophils of LAD-1 represents a distinct pathology from the commonly encountered forms of inflammatory bowel disease (IBD). The existence of active IBD in the presence of dysfunctional CD18/CD11(a-b) intercellular adhesion molecules (ICAM-1) interaction is relevant to the proposed targeting of ICAM-1 for the treatment of Crohn's disease.

Fibrinogen induces IL-8 synthesis in human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine or leukotriene B(4).

Human exudative neutrophils have greatly increased stores of the neutrophil chemoattractant IL-8 compared with peripheral blood cells, but the mechanism for the increase is not defined. In this report, we show that treatment of peripheral blood neutrophils with the chemotactic peptide fMLP or with leukotriene B(4) or fibrinogen results in little increase in the production of IL-8 by peripheral blood neutrophils. However, a chemotactically active dose of fMLP (5 x 10(-9) M) or leukotriene B(4) (1 x 10(-7) M) in the presence of a physiological concentration (2 mg/ml) of fibrinogen results in a receptor-mediated, pertussis toxin-sensitive, synergistic 30-fold increase in IL-8 synthesis. The levels of IL-8 attained are comparable to those observed in exudative cells. Higher concentrations of fMLP (1 x 10(-7) M) are associated with reduced IL-8 protein synthesis without IL-8 degradation, indicating a sensitive regulatory mechanism for IL-8 production. Treatment of neutrophils with fibrinogen and fMLP resulted in minimal changes in the steady state levels of mRNA for macrophage inflammatory protein-1alpha and -1beta and monocyte chemoattractant protein-1. In contrast, in the presence of fibrinogen, the steady-state level of neutrophil IL-8 mRNA increased 8-fold with 5 x 10(-9) M fMLP but was not decreased with 1 x 10(-7) M fMLP, suggesting that neutrophils are specifically adapted to modulate neutrophil IL-8 synthesis through transcriptional and posttranscriptional mechanisms. The data indicate that fibrinogen can function not only as a substrate in the clotting cascade, but also as an important effector during the evolution of the innate immune response.

Ca2+-dependent production and release of IL-8 in human neutrophils.

IL-8, a potent neutrophil chemoattractant that is elevated about 200-fold in exudative neutrophils isolated from localized inflammatory sites in vivo, is thought to play a major role in recruitment of neutrophils to inflammatory sites. Incubation of peripheral blood neutrophils with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-sequestering-ATPase, causes a dose-dependent induction of IL-8 synthesis that continues for up to 8 h. Cycloheximide inhibits the thapsigargin-induced IL-8 production, suggesting the induction of protein synthesis de novo. In addition, Northern blot analysis of mRNA isolated from neutrophils indicates that thapsigargin treatment increases IL-8 mRNA in a time- and dose-dependent manner. Thapsigargin also induces a biphasic rise in the intracellular Ca2+ concentration, [Ca2+]i, which is composed of an initial (within 15 s) EGTA-insensitive elevation in [Ca2+]i, followed by a delayed (2-min) EGTA-sensitive component. Addition of EGTA before thapsigargin inhibited the induction of IL-8 production. Experiments in which EGTA was added at various times after thapsigargin treatment indicated that a sustained Ca2+ influx was required for maximum IL-8 production. Ascomycin and cyclosporin A, inhibitors of the Ca2+-dependent phosphatase, calcineurin, also inhibited thapsigargin-induced IL-8 production. Thus, in neutrophils, a prolonged increase in [Ca2+]i stimulates IL-8 transcription and synthesis, possibly through a calcineurin-dependent pathway.

Monocyte chemotactic protein-2 (MCP-2) uses CCR1 and CCR2B as its functional receptors.

Monocyte chemotactic protein (MCP)-2 is a member of the C-C chemokine subfamily, which shares more than 60% sequence homology with MCP-1 and MCP-3 and about 30% homology with macrophage inflammatory protein (MIP)-1alpha, regulated on activation of normal T cell expressed (RANTES), and MIP-1beta. Despite this considerable sequence homology to other C-C chemokines, MCP-2 appears to have unique functional properties in comparison with other C-C chemokines such as MCP-1 and MCP-3. Although evidence obtained from studies on leukocytes suggested that MCP-2 may share the receptors with these C-C chemokines, the actual functional receptors for MCP-2 have not yet been identified. In this study, by using radioiodinated MCP-2, we identified high affinity binding sites for MCP-2 on human peripheral blood monocytes. The MCP-2 binding was competed for by MCP-1 and MCP-3, but less well by MIP-1alpha or RANTES. In experiments using cells transfected with C-C chemokine receptors, 125I-MCP-2 bound to human embryonic kidney 293 cells transfected with CCR1 or CCR2B, known to also bind MIP-1alpha/RANTES and MCP-1, respectively, but both shared by MCP-3. The binding of 125I-MCP-2 to these receptor-transfected cells was displaced completely by chemokines that bind to these receptors. Both CCR1- and CCR2B-transfected 293 cells showed significant migration in response to MCP-2, in addition to responding to other specific chemokines. These results clearly demonstrate that MCP-2, unlike MCP-1, uses both CCR1 as well as CCR2B as its functional receptors, and this accounts for the unique activities of MCP-2.

Endotoxin and IL-1 hyporesponsiveness in a patient with recurrent bacterial infections.

We describe a 15-yr-old girl with recurrent bacterial infections who is refractory to the effects of LPS in vivo and in vitro and IL-1 in vitro. Intravenous challenge of the patient with Escherichia coli endotoxin caused a subnormal febrile response, little alteration in the number of circulating neutrophils, and subnormal elevations in the plasma levels of TNF-alpha, IL-6, IL-8, lactoferrin, and granulocyte CSF; however, normal levels of the anti-inflammatory mediators IL-1 receptor antagonist and soluble TNF receptor (60 kDa) were induced. Studies in vitro indicated the patient's monocytes expressed CD14, the LPS receptor, and bound LPS in a specific manner, but failed to produce TNF-alpha and granulocyte CSF after stimulation with LPS, and failed to respond to IL-1, heat-killed Staphylococcus aureus, and soluble glucan. Peripheral blood patient neutrophils exhibited normal expression of CD14, but failed to respond to treatment with LPS (100-1000 ng/ml for 30 min at 37 degrees C), a treatment that caused increased expression of the surface markers, C10, CD18, CD11b, CD67, and CD45, and decreased expression of L-selectin in normal neutrophils. Treatment of normal and patient neutrophils with FMLP (0.1 microM) resulted in equivalent altered expression of these surface markers. Patient neutrophils could not be primed by either LPS or IL-1beta for enhanced FMLP-induced O2- generation, but primed normally to TNF-alpha and platelet-activating factor. This patient's hyporesponsiveness to LPS and IL-1 is most likely due to a defect very early in the signal-transduction pathway.

Interleukin 12 primes macrophages for nitric oxide production in vivo and restores depressed nitric oxide production by macrophages from tumor-bearing mice: implications for the antitumor activity of interleukin 12 and/or interleukin 2.

Interleukin-12 (IL-12) is a recently described immunoregulatory cytokine with potent therapeutic activity in various preclinical models of infectious or malignant disease. As part of our ongoing evaluation of potential mechanisms accounting for the potent antitumor activity of IL-12, we have investigated the influence of IL-12 administration on total serum nitrate/nitrite (NO(x)(-)) levels and the production of nitric oxide (NO) by peritoneal macrophages from normal and tumor-bearing mice. We report here that IL-12 administration to either normal or tumor-bearing mice for periods of time ranging from 7-19 days induced progressive increases in serum NO(x)(-) levels and primed peritoneal macrophages for NO production on subsequent exposure to lipopolysaccharide or IL-2 ex vivo. Treatment of resident peritoneal macrophages or the macrophage cell line ANA-1 with IL-12 alone or IL-12 in combination with various other stimuli failed to induce NO production, suggesting that the effects of IL-12 occurred via an indirect mechanism. Furthermore, we have shown that not only was the production of NO by macrophages from untreated long-term, tumor- bearing mice suppressed compared with control mice treated with vehicle or IL-12, but also that IL-12 administration overcame this suppression and delayed tumor growth. Lastly, we have shown that administration of weekly pulses of IL-2 in combination with IL-12 additively enhanced the priming of macrophages for NO production ex vivo and delayed tumor growth far more effectively than either agent alone. These observations and reports in the literature regarding the potential influence of NO on development of the immune response and on the regulation of tumor growth and vascularization suggest that NO may play a significant role in the antitumor activity of IL-12 and IL-2.

Monocyte chemotactic protein-3 (MCP3) interacts with multiple leukocyte receptors: binding and signaling of MCP3 through shared as well as unique receptors on monocytes and neutrophils.

The diversity of monocyte chemotactic protein (MCP)3 target cell types, as well as the capacity of MCP3 to desensitize leukocyte responses to other CC chemokines, suggested that MCP3 may interact with multiple CC chemokine receptors. The purpose of this study is to establish how MCP3 binds and activates monocytes and neutrophils. We show that human monocytes exhibit high-affinity binding for 125I-MCP3 with an estimated Kd of 1-3 nM and about 10,000 binding sites/cell. The binding of 125I-MCP3 to monocytes was progressively less well competed by CC chemokines macrophage inflammatory protein (MIP)1 alpha (Kd = 5-10 nM), RANTES (Kd = 5-10 nM), MCP1 (monocyte chemoattractant and activating factor, or MCAF) (Kd = 60 nM) and MIP1 beta (Kd > 100 nM). On the other hand, unlabeled MCP3 displaced the binding of radiolabeled MIP1 alpha, RANTES, MCP1 and MIP1 beta as effectively as the isologous CC chemokines. In agreement with the binding data, pretreatment of monocytes with MCP3 completely desensitized the calcium flux in response to MIP1 alpha and RANTES. However, MIP1 alpha and RANTES failed to desensitize the response of monocytes to MCP3. MCP3 and MCP1 partially desensitized each other's effects on monocytes. These binding and cross-desensitization results suggest that MCP3 binds and signals through other binding sites in addition to those shared with MIP1 alpha, RANTES and MCP1. The unidirectional competition for MIP1 beta binding and signaling by MCP3 suggests the existence of an as-yet unidentified site for MCP3 shared with MIP1 beta. The existence of another unique binding site(s) for MCP3 was further shown by the failure of any of the other CC chemokines to compete effectively for MCP3 binding on neutrophils. MCP3 in our study was also the only human CC chemokine that consistently chemoattracted neutrophils. These results suggest that MCP3 is a ligand that can bind and activate a broad range of target cells through receptors shared by other CC chemokines as well as its own receptor.

Loss of L-selectin (CD62L) on human neutrophils following exudation in vivo.

L-selectin, an adhesion molecule expressed on the surface of peripheral blood neutrophils, mediates the rolling of neutrophils along vascular endothelium. Stimulation of neutrophils in vitro causes decreased expression of L-selectin on the surface of neutrophils due to shedding. In this study, we have demonstrated that human exudative neutrophils isolated from both skin lesions and from pus exhibit little expression of L-selectin. Although a dramatic accumulation of exudative neutrophils is observed in the skin lesions within 24 hr, there is no accompanying increase in soluble L-selectin. In addition, the levels of soluble L-selectin in the extracellular tissue fluid (97.2 +/- 12.7 micrograms/ml, n = 8) are only 20% of that observed in peripheral plasma (482.4 +/- 35.9 micrograms/ml). These data suggest that shedding of L-selectin from the surface of neutrophils occurs in the peripheral circulation as a prelude to diapedesis of neutrophils into peripheral tissues.

Increased cell-associated IL-8 in human exudative and A23187-treated peripheral blood neutrophils.

IL-8 is a potent neutrophil chemoattractant that has been detected in high concentrations at acutely inflamed sites in vivo. Many cell types, including peripheral blood neutrophils, produce IL-8 that can be released by a variety of pro-inflammatory stimuli. However, the functional importance of neutrophil IL-8 during exudation is not yet known. We now report that neutrophils, harvested from skin lesions on the forearms of normal human volunteers (exudative neutrophils), expressed 100-fold higher levels of cell-associated IL-8 and spontaneously released up to 50-fold more IL-8 than freshly isolated peripheral blood neutrophils from the same donor. Furthermore, cell-associated IL-8 in peripheral blood neutrophils increased 20-fold during incubation at 37 degrees C in vitro and was increased over 200-fold after treatment with the Ca2+ ionophore A23187. More than 35% of the cell-associated IL-8 could be released by stimulation with either Ca2+ ionophore A23187 or phorbol myristate acetate. IL-8 was localized by sucrose gradient centrifugation to a subcellular fraction of heterogeneous, light membranous organelles. The accumulation of IL-8 within these organelles is inhibited by cycloheximide but not actinomycin D, suggesting that IL-8 accumulation is under translational, rather than transcriptional control. These studies indicate that peripheral blood neutrophils are capable of synthesis of large amounts of IL-8. Subsequent release of IL-8 during exudation may regulate neutrophil migration into sites of inflammation.

Increased circulating cytokines, cytokine antagonists, and E-selectin after intravenous administration of endotoxin in humans.

Intravenous administration of endotoxin into humans causes transient fever, alteration in the number of circulating neutrophils, and transient release into plasma of cytokines, cytokine antagonists, and other cellular products. The release can be temporally differentiated, and the extent of release is dose-dependent. By 1 h after endotoxin challenge, levels of tumor necrosis factor (TNF)-alpha and soluble TNF receptor increase; interleukin (IL)-6 and IL-8 increase by 1.5 h, and IL-1 receptor antagonist, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and lactoferrin increase by 2 h. Increased G-CSF is temporally associated with neutrophilia and the appearance of band neutrophils. Increased plasma lactoferrin and altered neutrophil surface antigen expression suggest that intravascular activation of neutrophils has occurred. The level of soluble E-selectin (sE-sel), an adhesion molecule released from endothelial cells, is elevated at 4 h and remains elevated at 24 h. sE-sel levels increase with higher doses of endotoxin at 4, 6, and 24 h.

Treatment of refractory disseminated nontuberculous mycobacterial infection with interferon gamma. A preliminary report.

BACKGROUND: Studies conducted in vitro and in animals suggest that cytokine signals to monocytes or macrophages by interferon gamma are important in the containment and clearance of disseminated nontuberculous mycobacterial infections. METHODS: We studied seven patients with refractory, disseminated nontuberculous mycobacterial infections who were not infected with the human immunodeficiency virus. Three patients were from a family predisposed to the development of Mycobacterium avium complex infections; four patients had idiopathic CD4+ T-lymphocytopenia. Their infections were culture- or biopsy-proved, involved at least two organ systems, and had been treated with the maximal tolerated medical therapy. Cellular proliferation, cytokine production, and phagocyte function were assessed in peripheral-blood cells. Interferon gamma was administered subcutaneously two or three times weekly in a dose of 25 to 50 micrograms per square meter of body-surface area in addition to antimycobacterial medications. Clinical effects were monitored by cultures, biopsies, radiographs, and in one patient a change in the need for paracentesis. RESULTS: In response to phytohemagglutinin, the production of interferon gamma by mononuclear cells from the patients was lower than in normal subjects (P < 0.001), whereas stimulation with ionomycin and phorbol myristate acetate led to normal production of interferon gamma in the patients. Within eight weeks of the start of interferon gamma therapy, all seven patients had marked clinical improvement, with abatement of fever, clearing of many lesions and quiescence of others, radiographic improvement, and a reduction in the need for paracentesis. CONCLUSIONS: Interferon gamma in combination with conventional therapy may be effective for some cases of refractory disseminated nontuberculous mycobacterial infection.

Structure and functional expression of the human macrophage inflammatory protein 1 alpha/RANTES receptor.

The chemokine beta family is comprised of at least six distinct cytokines that regulate trafficking of phagocytes and lymphocytes in mammalian species; at least one of these, macrophage inflammatory protein 1 alpha (MIP-1 alpha), also regulates the growth of hematopoietic stem cells. We now show that MIP-1 alpha and the related beta chemokine, RANTES, induce transient alterations in intracellular Ca2+ concentration in polymorphonuclear leukocytes that can be reciprocally and specifically desensitized, suggesting a common receptor. Moreover, we have now cloned both the cDNA and the gene for this receptor, functionally expressed the receptor in Xenopus oocytes, and mapped the gene to human chromosome 3p21. Transcripts for the receptor were found in mature and immature myeloid cells as well as B cells. The receptor is a member of the G protein-coupled receptor superfamily. It has approximately 33% amino acid identity with receptors for the alpha chemokine, interleukin 8, and may be the human homologue of the product of US28, an open reading frame of human cytomegalovirus.

Dynamics of the cellular and humoral components of the inflammatory response elicited in skin blisters in humans.

Skin blisters induced by suction on the forearm of normal volunteers provide a convenient model to study the inflammatory response in vivo in man. In our study, after removal of the roof of the blister, i.e., the epidermis, the exposed floor of the blister (dermal-epidermal interface) was bathed with 70% autologous serum using a multiwell skin chamber. Migration of leukocytes (90-95% neutrophils) into the chamber fluid was detectable within 3 h, and appeared to plateau at 16-24 h. Sampling of the dermal-epidermal interface revealed primarily mononuclear cells during the first 8 h of the inflammatory response; however, their prevalence at 24 h was greatly diminished due to neutrophil infiltration. Accompanying the cellular immune response was the accumulation of inflammatory mediators in the bathing medium. The accumulation of IFN-gamma reached a plateau within 3 h; significant accumulations of the complement fragment, C5a, and of leukotriene B4 were also detected at 3 h. The accumulation of C5a did not peak until 5 h, whereas leukotriene B4 continued to accumulate through 24 h. IL-6 and IL-8 concentrations were minimal at 3-8 h but dramatic by 24 h while IL-1 beta, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor were undetectable within 3-8 h, but markedly elevated by 24 h. There was little accumulation of IL-4 and no accumulation of IL-1 alpha or IL-2 during the 24-h period. The sequential appearance of mediators at an inflammatory focus suggests that a carefully regulated dynamic system is responsible for controlling the evolution of the inflammatory response.

A role for protein tyrosine kinase activity in natural cytotoxicity as well as antibody-dependent cellular cytotoxicity. Effects of herbimycin A.

NK cells, CD3- large granular lymphocytes, have diverse means by which they lyse targets, including antibody-dependent cellular cytotoxicity. The low affinity receptor for the Fc portion of Ig (Fc gamma RIIIA), like the TCR, is a multimeric receptor complex coupled to a protein tyrosine kinase. In the present study, we observed that inhibition of tyrosine kinase activity by herbimycin A interferes with receptor-mediated phosphorylation of a variety of substrates and mobilization of intracellular calcium. Fc gamma RIIIA induced IL-2R alpha-chain expression was also extremely sensitive to herbimycin A as was antibody-dependent cellular cytotoxicity, in fact more so than receptor-mediated phosphorylation and calcium mobilization. In contrast to Fc gamma RIIIA, the surface molecules and biochemical mechanisms involved in NK cytotoxicity and lymphokine-activated killing are not well characterized. Interestingly, however, herbimycin A also blocks these modes of cytolysis, implicating a role for tyrosine kinase function in these processes. Whether FcR-mediated signaling and receptor-mediated signaling involved in NK activity share specific biochemical intermediates is not known, but the involvement of tyrosine kinase function in the latter means of cytotoxicity may provide novel avenues for understanding the biochemical basis of this perplexing cellular function.

Anti-IgM-mediated growth inhibition of a human B lymphoma cell line is independent of phosphatidylinositol turnover and protein kinase C activation and involves tyrosine phosphorylation.

The RL cell line is an EBV-negative, surface IgM, IgD-positive B lymphoma line, which is significantly growth arrested in the presence of acrylamide-linked antibodies to the surface IgM receptor. We demonstrate here that activation of protein kinase C (PKC) with PMA abrogates anti-IgM-induced phosphoinositide turnover and Ca2+ mobilization; however, growth inhibition is not affected. In addition, inhibitors of PKC are unable to reverse the anti-IgM-mediated growth inhibition. Two-dimensional gel electrophoresis reveals a different pattern of protein phosphorylation after treatment of RL with PMA or anti-IgM. These data strongly suggest that anti-IgM-induced growth inhibition does not rely on phospholipase C-mediated phosphoinositide turnover, Ca2+ mobilization, or PKC activation. On the other hand, the phosphatase inhibitor orthovanadate results in an augmentation of proteins phosphorylated on tyrosine and the growth inhibition which follows anti-IgM treatment. Furthermore, protein tyrosine kinase inhibitors, genistein and herbimycin A, are able to reverse the anti-IgM-induced inhibition of growth. These data demonstrate that multiple signaling pathways are activated by the interaction of anti-IgM with its ligand, and suggest that tyrosine kinase activation is a critical component of the inhibitory response.

Endothelial cells inhibit receptor-mediated superoxide anion production by human polymorphonuclear leukocytes via a soluble inhibitor.

Confluent monolayers of bovine pulmonary artery endothelial cells (BPAE) or human umbilical vein endothelial cells (HUVE) inhibited by 80 to 90% the production of O2- by added human neutrophils (PMNs) stimulated by plasma membrane receptor-mediated activators (formylmethionylleucylphenylalanine [fMLP], opsonized zymosan, heat-killed Staphylococci), but not by non-plasma membrane receptor-mediated activators (phorbol myristate acetate and delta-hexachlorocyclohexane). Degranulation induced by fMLP was also inhibited by BPAE. Inhibition was not affected by eicosatetraynoic acid (ETYA) or indomethacin. To assess the role of cell-cell contact, 0.45-microns-pore culture plate inserts were employed to prevent PMN-endothelial cell contact during incubation. A similar amount of inhibition of stimulated PMNs superoxide production was seen as compared to PMN-endothelial incubations where contact occurred. A soluble component released by BPAE monolayers, when added to PMNs, duplicated the inhibition seen by BPAE-PMN co-incubation. Incubation of BPAE with adenosine deaminase did not reduce inhibition of O2- production compared to controls without adenosine deaminase. There was no evidence of endothelial scavenging of O2- generated by hypoxanthine-xanthine oxidase, and inhibition of endothelial superoxide dismutase did not diminish the inhibitory effort. We conclude that cell contact is not required for BPAE inhibition of fMLP-stimulated O2- production by PMN, and that scavenging of superoxide anion is not the mechanism. The inhibitor appears to be a polypeptide with an apparent molecular weight between 1,000 and 10,000 D and does not appear to be adenosine, an arachidonate metabolite, or superoxide dismutase. The mechanism may involve down-regulation of plasma membrane receptor-mediated activation of PMNs.

Chondrocyte activation by a putative interleukin-1 derived from lapine polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) recovered from 4-h lapine peritoneal exudates contained factors which provoked the synthesis of collagenase, gelatinase, caseinase, and prostaglandin E2 by lapine articular chondrocytes. Rapid secretion of these factors occurred after exposing the polymorphs to phorbol myristate acetate or formyl-Met-Leu-Phe. Fractionation of polymorph lysates by HPLC size exclusion chromatography provided a molecular weight of approximately 14,000 for the active principle. Examination of the most active fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining, confirmed the presence of a single band with this apparent molecular weight. Isoelectric focusing of this fraction revealed the presence of four distinct bands with the pI values 6.90, 7.05, 7.45, and 7.55. This fraction tested positive in a bioassay for interleukin-1. We were unable to activate chondrocytes by exposure to extracts of human PMNs from either peripheral blood or inflammatory synovial fluid.

Essential fatty acid deficiency and neutrophil function: studies of lipid-free total parenteral nutrition in monkeys.

Linoleic acid (18:2 omega 6), the obligate precursor of arachidonic acid (20:4 omega 6), is an essential nutrient for humans and other mammals. Because arachidonic acid release and metabolism are components of neutrophil activation by certain stimuli, we questioned whether neutrophils depleted of arachidonate as a result of essential fatty acid deficiency might be functionally impaired. We examined this possibility by producing essential fatty acid deficiency in monkeys with lipid-free total parenteral nutrition (TPN). Rhesus and African green monkeys were given calorically equal TPN for up to 23 days with and without a vegetable fat emulsion rich in linoleic acid. Fatty acids were analyzed in total lipid extracts of serum and isolated blood neutrophils by gas-liquid chromatography. Although fatty acids in the serum and neutrophils of monkeys given TPN with lipid did not change, linoleic acid levels decreased by at least 60% in serum and 50% in neutrophils from animals given TPN with no lipid. Moreover, arachidonate levels in neutrophil lipids decreased by at least 50% within 12 days of lipid-free TPN, and the abnormal fatty acid 20:3 omega 9 (characteristic of essential fatty acid deficiency) appeared and steadily increased with time. These biochemical signs of omega 6 fatty acid deficiency were associated with impaired neutrophil function in vitro. Both migration responses and superoxide generation stimulated by N-formyl-methionyl-leucyl-phenylalanine were significantly decreased by 12 days of lipid-free TPN, as was the capacity of activated cells to synthesize leukotriene B4. In contrast, functional responses of fatty acid-deficient neutrophils to leukotriene B4 and phorbol myristate acetate, which have little or no effect on arachidonate release or metabolism, were not significantly altered. These findings indicate that endogenous supplies of arachidonic acid and other essential omega 6 fatty acids influence the functional responsiveness of neutrophils. These studies also indicate that altered neutrophil function is a feature of essential fatty acid deficiency and that it may contribute to the increased risk of infection and decreased inflammatory responses observed in this condition.

ATP induces transient elevations of [Ca2+]i in human neutrophils and primes these cells for enhanced O2- generation.

ATP, when added to human polymorphonuclear neutrophils (PMNs) at concentrations similar to those attained extracellularly at sites of platelet thrombus formation (0.1 to 20 microM), causes an enhancement of N-formyl(methionyl)leucylphenylalanine (FMLP)-stimulated superoxide anion (O2-) generation. However, ATP by itself is an ineffective agonist for O2- generation by PMNs. The ATP-induced enhancement of O2- generation is associated with a shortened lag time in the response of PMNs to FMLP without a change in the median effective dose for FMLP, suggesting that signal transduction, rather than altered receptor affinity, is responsible for the enhanced oxidative response. Maximum enhancement of O2- generation is detected as early as 15 seconds and is maintained for at least 10 minutes. Of various nucleotides and nonhydrolyzable-ATP analogs test d, only ATP, UTP, and ITP were found to cause enhanced O2- generation by PMNs. Addition of ATP to quin2-loaded PMNs, in the absence of other stimuli, elicits a dramatic rise in [Ca2+]i which reaches a maximum of 500 to 800 nM at 30 seconds and slowly returns to baseline over 5 minutes. This ATP-induced rise in intracellular free Ca2+ concentration is correlated with the enhancement of FMLP-stimulated O2- generation both with respect to dose and nucleotide specificity. Stimulated Ca2+ uptake, rather than mobilization of intracellular Ca2+ stores, appears to be primarily responsible for the rise in intracellular free Ca2+ concentration. These studies indicate that an ATP-induced rise in intracellular free Ca2+ concentration, although insufficient by itself to elicit O2- generation by PMNs, is associated with a priming of PMNs for enhanced O2- generation when stimulated by other agonists.

Inhibition of cell movement and associated changes by hexachlorocyclohexanes due to unregulated intracellular calcium increases.

Hexachlorocyclohexanes (HCCHs) are potent stimulators of polymorphonuclear leukocyte (PMN) oxidative metabolism and of mobilization of calcium from intracellular stores. It was of interest, therefore, to evaluate the effect of HCCHs on PMN orientation and chemotaxis and to determine their effectiveness as chemotaxins. Chemotaxis was evaluated using micro-Boyden chambers, f-actin was quantitated by nitrobenzoxadiazole (NBD)-phallacidin fluorescence, and microtubules were quantitated by observing the concanavalin A (Con A) capping phenomenon. We also evaluated changes in intracellular calcium [Ca2+]i using quin 2 fluorescence. We found that the HCCH isomers were not chemotaxins and that the HCCH isomers that stimulated O2- formation (delta and gamma HCCH) inhibited chemotaxis. This effect was associated with inhibition of orientation. In addition, we found extensive inhibition of both f-actin and Con A cap formation. These effects of HCCH on cell function were associated with marked increases of [Ca2+]i. This work suggests that non-receptor-mediated increases of [Ca2+]i associated with HCCH have divergent effects on cell function and suggests that physiologic responses of PMNs requiring cytoskeletal alterations, such as chemotaxis, depend on the controlled responses of receptor-mediated stimulation.