Immunoglobulin G-induced single ionic channels in human alveolar macrophage membranes.

While it is well known that the engagement of IgG Fc receptors on the macrophage surface triggers a number of cellular responses, including particle ingestion, secretion, and respiratory burst activity, the mechanism of signal transmission following ligand binding remains poorly understood. To acquire more data in this area, we studied the electrical properties of the macrophage membrane and its response to oligomeric immunoglobulin G (IgG) using the patch-clamp technique on human alveolar macrophages that were obtained by bronchoalveolar lavage and maintained in short-term tissue culture. The results showed that cell resting potentials, as determined from whole-cell tight seal recordings, increased from -15 mV on the day of plating to -56 mV after the first day in culture and remained stable at this hyperpolarized level. Macrophages revealed an input resistance of 3.3 G omega, independent of age in culture. Extracellular application of heat-aggregated human IgG to cells voltage-clamped at -70 mV resulted in peak inward currents of approximately 470 pA. We identified an IgG-dependent, nonselective channel in both cell-attached and isolated membrane patches, with a unitary conductance of approximately 350 pS and a predominant subconductance level of 235 pS in symmetrical NaCl solutions. Single channel open times were observed to be in the range of seconds and, in addition, were dependent upon membrane voltage. Channel opening involved transitions between a number of kinetic states and subconductance levels. Channel events recorded in cell-attached patches showed characteristic exponential relaxations, which implied a variation in membrane potential as a result of a single ion channel opening. These data suggest that the IgG-dependent nonselective cation channel that we have characterized may provide the link between Fc receptor engagement and subsequent cellular activation.

C-reactive protein selectively enhances the intracellular generation of reactive oxygen products by IgG-stimulated monocytes and neutrophils.

The acute phase protein, C-reactive protein (CRP), when heat-aggregated (Agg-CRP), potentiates immunoglobulin G (IgG) Fc receptor-mediated luminol-enhanced chemiluminescence (CL) in human monocytes and neutrophils. Luminol-CL is a sensitive measure of phagocyte respiratory burst activity; however, the nature of oxidative products contributing to the light emission and their site of generation remain incompletely defined. To more precisely describe the oxidative burst of monocytes and neutrophils to Agg-CRP, superoxide anion release was measured by cytochrome c reduction. In addition, the extracellular release of hydrogen peroxide was distinguished from hydrogen peroxide generation using a phenol red oxidation assay. Finally, a flow cytometric determination of dichlorofluorescein (DCFH) oxidation was employed as an index of intracellular peroxide production. Although Agg-CRP alone did not stimulate hydrogen peroxide generation by either monocytes or neutrophils, it significantly enhanced hydrogen peroxide generation in response to heat-aggregated IgG (Agg-IgG). In contrast, Agg-CRP did not enhance the extracellular release of either hydrogen peroxide or superoxide anion from Agg-IgG-stimulated cells. The capacity of Agg-CRP to enhance selectively intracellular oxidative product generation was confirmed when measuring DCFH oxidation in Agg-IgG-stimulated cells. To evaluate whether this selective enhancement of intracellular oxidative events could be attributed, at least in part, to a scavenging effect of Agg-CRP, a cell-free oxygen radical-generating system was employed. Agg-CRP did not significantly diminish the lucigenin-amplified CL response induced by the xanthine/xanthine oxidase reaction. These results indicate that although Agg-CRP enhances the intracellular generation of reactive oxygen intermediates by monocytes and neutrophils, extracellular release of those products is not influenced by cell interaction with Agg-CRP. It is tempting to speculate that CRP can selectively boost the microbicidal activities of monocytes and neutrophils within an inflammatory site by amplifying the intracellular generation of reactive oxygen products without increasing damage to surrounding normal tissues.

Functional resistance of inflammatory macrophages to methotrexate in vitro.

The purpose of this study was to evaluate the effects of high and low therapeutic doses of methotrexate (MTX) on macrophage metabolism and function in vitro. Monolayers of elicited rat peritoneal macrophages (PM) were exposed to a wide range of MTX concentrations (10(-8) M-10(-3) M) for 24 or 48 hr and macrophage RNA and protein metabolism were evaluated by the incorporation of [3H]5-uridine and [14C]1-leucine, respectively, into trichloroacetic acid (TCA)-precipitable material. Macrophage functional activity was examined by measuring the uptake of [14C]Pseudomonas aeruginosa to assess phagocytosis and the release of 51Cr from antibody-coated [51Cr]sheep red blood cells (SRBC) to assess antibody-dependent cell-mediated cytotoxicity. Following a 24-hr incubation with 10(-3) M MTX, incorporation of [3H]5-uridine into PM monolayers was enhanced 79% when compared to control monolayers (p less than 0.005). Washout studies revealed that the stimulation of uridine incorporation was no longer observed by 24 hr following the removal of MTX from the culture medium. Incubation with 10(-3) M MTX for 48 hr returned uridine incorporation to control levels, although leucine incorporation into protein was reduced by 22% (p less than 0.01). The depression in leucine incorporation in the presence of 10(-3) M MTX was not reversed after the removal of MTX from the culture medium. Uptake of [14C]P. aeruginosa was not altered following a 24- or 48-hr incubation with either 10(-7) M or 10(-3) M MTX. Similarly, [51Cr]SRBC cytolysis was not affected by a 2-hr preincubation with and continuous exposure to between 10(-8) M and 10(-3) M MTX. These results demonstrate that incubation of inflammatory macrophages with clinically high doses of MTX can alter macrophage RNA and protein metabolism without producing demonstrable changes in macrophage functional activity.

Enhancement of human peripheral blood monocyte respiratory burst activity by aggregated C-reactive protein.

We had previously demonstrated that C-reactive protein (CRP), an acute phase reactant, when aggregated or coupled to a ligand, interacts with monocytes and certain human peripheral blood lymphocytes. The purpose of the present study, after further characterizing the binding interaction of CRP with human monocytes, was to focus on the biological response of monocytes to CRP binding. Flow cytometric analysis of human mononuclear leukocytes, following incubation with fluoresceinated heat-aggregated CRP (Agg-CRP), revealed that while greater than 70% of monocytes bound Agg-CRP, only 8% of lymphocytes demonstrated positive fluorescence. Furthermore, mean channel fluorescence values indicated that monocytes bound greater amounts of Agg-CRP per cell than did lymphocytes. Luminol-enhanced chemiluminescence (CL) was used as a measure of monocyte respiratory burst activity. Monocytes were stimulated only minimally by Agg-CRP alone; however, Agg-CRP substantially enhanced the CL response to heat-aggregated IgG. This Agg-CRP enhancing effect was selective for IgG-initiated monocyte activation, as no augmentation in CL was observed following cell stimulation with phorbol myristate acetate or serum-opsonized zymosan. These results demonstrate that aggregated CRP binds to the major proportion of human monocytes and selectively augments Fc receptor-mediated stimulation of monocyte oxidative metabolism.

Enhancement of human monocyte and peritoneal macrophage chemiluminescence activities in women with endometriosis.

Preliminary reports indicate that products of human mononuclear phagocytes may contribute to the infertility associated with endometriosis. To determine whether the generation of reactive oxygen metabolites by blood monocytes and peritoneal macrophages is altered in women with endometriosis, the present study evaluated luminol-enhanced chemiluminescence (CL) in cells at rest and following stimulation with phorbol myristate acetate (PMA) or serum-opsonized zymosan (SOZ). Peripheral venous blood and peritoneal fluid samples were collected from 60 infertile women undergoing diagnostic laparoscopy at midluteal phase and mononuclear phagocytic cell fractions were obtained by density gradient centrifugation. Whereas there was no significant difference between resting CL values in peripheral blood monocytes collected from women with and without endometriosis, PMA- and SOZ-stimulated monocyte CL was significantly greater in endometriosis patients. In contrast, there was a significant elevation in resting CL values when peritoneal macrophages from endometriosis patients were compared with macrophages obtained from patients with normal pelvic organs. It appears that chronic stimulation of macrophages in the peritoneal cavity provokes constitutive release of large quantities of reactive oxygen products in women with endometriosis. This may occur secondary to the accumulation of activated monocytes into the peritoneal cavity.

Changes in respiratory burst activity during human monocyte differentiation in suspension culture.

Monocytes undergo a process of differentiation following their accumulation into extravascular spaces. This process has been examined previously by culturing monocytes and identifying changes in cell morphology, metabolism, and function over time. The present study was designed to characterize mononuclear phagocyte respiratory burst activity as related to differentiation by measuring chemiluminescence and superoxide anion generation in cultured human monocytes. Monocytes maintained in Teflon vials for up to 12 days increased in size, were positive for nonspecific esterase, and retained the ability to ingest latex particles. During culture, however, cells progressively lost their peroxidase-positive granules. When monocytes were cultured for one or five days, they elicited less than 50% of the luminol-enhanced chemiluminescence produced by fresh monocytes following PMA stimulation. By day 7, less than 20% of day 0 PMA-elicited chemiluminescence was observed. A comparable loss of serum-opsonized zymosan-induced chemiluminescence occurred during monocyte culture. Since it is recognized that luminol-enhanced chemiluminescence is, in large part, dependent upon myeloperoxidase and since differentiated mononuclear phagocytes are only minimally peroxidase-positive, cultured monocyte respiratory burst activity was also assessed by directly quantifying superoxide anion generation. When monocytes were cultured for three or five days, they elicited 38% more superoxide anion than did fresh monocytes following PMA stimulation. At day 7, PMA-induced superoxide anion release was comparable to day 0 levels. These data indicate that monocytes allowed to differentiate under nonadherent conditions maintain the ability to undergo a respiratory burst response as measured by superoxide anion release, but they concomitantly lose peroxidase-dependent luminol-enhanced chemiluminescence. In this regard, monocytes cultured in suspension metabolically resemble macrophages that have undergone differentiation within sites of inflammation.

Effects of high-dose methotrexate on rat alveolar and inflammatory macrophage populations.

Methotrexate (MTX) is used clinically in high doses to treat a variety of neoplastic diseases. Although it has been recognized that such aggressive chemotherapy can result in suppression of cellular host defense mechanisms, phagocytic cell function during MTX therapy remains poorly understood. The present study was designed to examine the effects of a single high dose of MTX on alveolar and inflammatory peritoneal cell populations in the rat. Male Sprague-Dawley rats were treated with a single intraperitoneal injection of MTX (25 or 50 mg/kg) and subsequent alterations in the composition of peripheral blood white cells (WBC), a peritoneal inflammatory exudate, and the resident alveolar macrophage (AM) were measured 1-4 days after MTX treatment. A severe depression in the polymorphonuclear leukocyte (PMNL) and monocyte populations in the peripheral blood was observed 72 and 96 h after either dose of MTX. Similarly, the total number of peritoneal exudate cells, collected 72 h after a caseinate inflammatory stimulus, was reduced by 50% after MTX treatment. When the cellular composition of the peritoneal exudate was examined, it was observed that macrophage and lymphocyte numbers were selectively depressed. In addition, the number of AM obtained by lung lavage was significantly reduced 72 and 96 h after MTX injection. Although both peritoneal and alveolar macrophage numbers were diminished, in vitro phagocytic activity was not impaired 72 h after MTX injection. These studies demonstrate that a single, clinically relevant dose of MTX, in addition to depressing PMNL and monocyte levels in the peripheral blood, can also impair the accumulation of macrophages at a site of tissue injury and the influx of macrophages into lung alveoli. These findings suggest that the capacity of the mononuclear phagocyte system to respond to an infectious or tumor cell challenge may be severely compromised during MTX treatment.

Stimulation of human neutrophils, monocytes, and platelets by modified C-reactive protein (CRP) expressing a neoantigenic specificity.

C-reactive protein (CRP) can be structurally modified by heat, acid, or urea-chelation to express a neoantigen designated by us as neo-CRP. This antigen is also expressed on the in vitro primary protein translation products of both human and rabbit CRP. Unmodified CRP and CRP complexed with pneumococcal C-polysaccharide (CPS) do not express neo-CRP. Forms of CRP expressing neo-CRP but not native CRP antigenicity (even in the presence of CPS) consistently and in a dose-dependent manner potentiated the respiratory burst response of human polymorphonuclear leukocytes and peripheral blood monocytes to heat-modified IgG. Forms of CRP expressing neo-CRP antigenicity also induced reactions of aggregation and secretion from isolated platelets and potentiated platelet activation stimulated by ADP in platelet-rich-plasma, while native CRP alone or complexed with CPS again did not. Unlike CRP-CPS complexes, forms of CRP expressing neo-CRP were not able to activate the complement system. These data emphasize the biologic potential inherent in this humoral acute-phase reactant, particularly in the activation of the formed elements of the blood important in the inflammatory response. Since these cell-activating properties are preferentially observed when CRP is structurally modified to express the neo-CRP antigen, such a molecular conversion may be central to the structure-function relationships of CRP at local sites of inflammation and tissue injury.

Suggestions for clinical nursing research: symptom management in AIDS patients.

Clinical nurse researchers are challenged to formulate and test effective interventions to alleviate the symptoms associated with HIV/AIDS. Although many symptom-management studies have been conducted on specific populations, such as oncology patients, few related nursing studies of persons infected with HIV have been done. The authors offer suggestions for studying symptom management approaches for persons with AIDS. The suggestions represent the funding priorities identified by the National Institute of Nursing Research.

Immunological and virological markers of HIV-disease progression.

This review, based upon scientific literature, evaluates a number of immunological and virological markers for their usefulness as prognostic indicators for progression of HIV disease. The most widely studied marker, the CD4 positive T lymphocyte count, is perhaps the best single indicator of stage of illness. Serum factors such as neopterin and beta-2 microglobulin, alone and in combination with CD4 cell counts, have been shown to have good predictive value. Measurement of viral burden by quantification of viral RNA levels in plasma and immune cells also holds promise for following disease progression. It is recommended that a combination of these factors be monitored in evaluating stage of illness and responses to therapy in HIV-infected persons.

HIV-associated distal symmetrical polyneuropathy: clinical features and nursing management.

DSPN is a common manifestation of HIV infection and/or its treatment that can have adverse effects on quality of life and functional status. The pathogenesis remains unclear but likely involves the elaboration of neurotoxic inflammatory cytokines and their metabolites. DSPN is often refractory to available pharmacological treatments, although new treatments involving NGF hold promise for effecting sustained symptom relief and reversing axonal degeneration. Further research is needed to determine the efficacy of nonpharmacological treatments, such as cognitive-behavioral therapies, to alleviate DSPN-associated pain.

Cortisol upregulates HIV p24 antigen production in cultured human monocyte-derived macrophages.

HIV infection is associated with hypercortisolemia. Since glucocorticoids have been shown to stimulate the replication of several viruses, we examined the effects of cortisol on HIV replication in cultured monocyte-derived macrophages (MDM), a cell type that has been proposed to serve as a viral reservoir. Our data revealed that physiological concentrations of cortisol upregulate viral replication in MDM. Because the dose-response curve for cortisol on HIV replication in vivo is not known, the clinical relevance of these findings remain uncertain. Clinical studies are needed to characterize the effects of corticosteroid therapy on viral burden in vivo.

Complementary and alternative therapies to manage HIV-related symptoms.

Persons with HIV infection report substantial use of complementary and alternative medical (CAM) therapies for symptom management. Anecdotal reports from patients indicate that CAM approaches are helpful; however, there is limited scientific information on the safety and efficacy of these therapies in the HIV population. The purpose of this review is to critically appraise the scientific evidence for selected CAM therapies that are used by HIV-infected persons to manage three common symptoms: nutritional alterations, pain, and depression.

Surgical implants. Physiological response.

Following the surgical insertion of all prosthetic implants, an identical sequence of events occurs, although the magnitude of each response is variable. Initially, to maintain hemostasis, the coagulation system is stimulated and platelet activation occurs. Within minutes, leukocytes accumulate in an attempt to neutralize the foreign body. Although these phagocytic cells are generally unsuccessful at breaking down the biomaterials comprising an implant, macrophages coalesce and wall-off the foreign body from surrounding tissues. Once the inflammatory response is quiescent, fibroblasts invade the granuloma, lay down connective tissue and contract to minimize the volume occupied by the implant. Although most implant patients experience uneventful recoveries, complications resulting from chronic activation of the inflammatory response or the mechanisms involved in maintaining hemostasis can occur.

Oropharyngeal administration of colostrum to extremely low birth weight infants: theoretical perspectives.

Studies in adults have shown that the oropharyngeal route can be used to effectively and safely administer interferon-alpha, an immune cell-derived cytokine, to patients who are unable to tolerate its parenteral administration. The mechanism for this appears to be the stimulatory effects of the cytokine, on the oropharyngeal-associated lymphoid tissue system. Own mother's colostrum (OMC) is rich in cytokines and other immune agents that provide bacteriostatic, bacteriocidal, antiviral, anti-inflammatory and immunomodulatory protection against infection. OMC may be especially protective for the extremely low birth weight (ELBW) infant in the first days of life; however clinical instability typically precludes enteral feedings during this period. Oropharyngeal administration is a potential alternative method of providing OMC. Oropharyngeal administration of OMC may have immunomodulatory effects on the recipient infant, and would be especially beneficial to the ELBW infant who would otherwise remain nil per os during the first days of life.

Monoclonal antibody to the type II Fc receptor for human IgG blocks potentiation of monocyte and neutrophil IgG-induced respiratory burst activation by aggregated C-reactive protein.

The acute phase protein, CRP, when heat-aggregated (Agg-CRP), binds to human monocytes and neutrophils and potentiates the respiratory burst stimulated by heat-aggregated IgG (Agg-IgG). Earlier data from our laboratory and others have indicated that CRP binds to phagocytic cells at membrane sites associated with IgG Fc receptors. The present study utilized monoclonal antibodies (MAb) to determine whether the Agg-CRP potentiation of oxidative metabolism could be linked to activation through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Preincubation of monocytes with MAb 32.2, which recognizes an Fc gamma RI epitope distinct from its IgG binding site, had only a minimal (20%) inhibitory effect on Agg-IgG-induced luminol chemiluminescence (CL) and exerted no significant effect on its enhancement by Agg-CRP. MAb 10.1, which blocks IgG binding to Fc gamma RI, reduced Agg-IgG-induced monocyte CL by 40%, but did not alter the Agg-CRP-mediated enhancement. In contrast, exposure to MAb IV.3, which binds to Fc gamma RII on monocytes and neutrophils and blocks IgG binding to this receptor, resulted in a greater than 70%, inhibition of Agg-IgG-induced CL and also significantly suppressed the enhancement by Agg-CRP. MAb Leu-11b, which reacts with Fc gamma RIII on neutrophils, reduced Agg-IgG-induced CL by 70% but did not suppress the Agg-CRP potentiation. Preincubation of monocytes and neutrophils with anti-Leu-M1, anti-CR1, or anti-CR3 failed to block Agg-IgG-induced CL or its enhancement by Agg-CRP. Although the potentiating effect of Agg-CRP on Agg-IgG-elicited CL was blocked by MAb IV.3, this antibody failed to reduce binding of Agg-CRP to either monocytes or neutrophils. These results indicate that, although Agg-CRP does not bind to phagocytic cells at the IgG-binding determinant of Fc gamma RII, it alters Agg-IgG-induced cell activation through this receptor.

Modulation of inflammatory peritoneal cell function and metabolism by methotrexate.

Methotrexate (MTX) is widely used in cancer chemotherapy, although the effects of MTX on cellular antitumor defense mechanisms are poorly understood. To evaluate the effect of MTX on the cellular inflammatory response, male Sprague-Dawley rats were treated with four daily i.p. injections of MTX or a control vehicle. Rats treated with daily doses of 1.2 mg/kg MTX demonstrated a significant reduction in number of peritoneal exudate cells, specifically macrophages, collected 96 hours following the inflammatory stimulus. To determine if metabolic perturbations also occur upon exposure to MTX, glucose oxidation and protein synthesis by inflammatory cells were monitored in vitro. At a MTX concentration of 10(-3)M, peritoneal exudate cell 14C-1-glucose and 14C-6-glucose oxidation was significantly depressed. 14C-1-leucine incorporation into TCA precipitable protein was inhibited at 4 x 10(-3)M MTX. Peritoneal exudate cell viability was not altered at these concentrations of MTX. These results demonstrate that MTX, at therapeutic concentrations, can depress the influx of macrophages to a inflammatory site and also diminish energy metabolism and protein synthesis by inflammatory cells.

Psychoneuroimmunology: an emerging framework for nursing research.

Psychoneuroimmunology (PNI) is concerned with the mechanisms of bidirectional communication between the neuroendocrine and immune systems. Investigators in other disciplines have used this framework to guide the examination of possible relationships between behavioural factors and the progression of immunologically mediated illnesses and to evaluate the role of immune products in central nervous system disturbances. Nurse scientists have an opportunity to make unique contributions to the growing field of PNI. Unlike basic science research, which has as its goal the generation of fundamental knowledge concerning biological or behavioural processes, nursing research is driven by the need to promote excellence in nursing science as a guide for nursing practice. Although a few nurse scientists have conducted PNI research to date, additional studies are needed to generate new knowledge concerning mind-body interactions in health and illness and to develop strategies that promote mental and physical well-being in persons at risk for immune dysfunction. This paper highlights the few recently conducted nursing studies grounded in a PNI framework to illustrate the utility of PNI in advancing nursing science.

Evaluation of human monocyte oxidative metabolism utilizing a flow cytometric assay.

Assays routinely employed to evaluate human monocyte respiratory burst activation have been limited to measuring responses of bulk cell preparations. We demonstrate that individual monocyte responses can be easily assessed by using 2',5' dichlorofluorescin diacetate (DCFH-DA) and flow cytometry. Adherence purified monocytes were incubated with DCFH-DA, washed, and stimulated with either phorbol myristate acetate (PMA) or heat-aggregated IgG (HAIgG). Log green fluorescence signals were measured by using a flow cytometer equipped with a 5-W argon laser set at an excitation wavelength of 488 nm. Optimal conditions for stimulation included exposure to 5 microM concentrations of DCFH-DA for 15 min, followed by a 60-min incubation with either PMA or HAIgG. Dichlorofluorescin (DCFH) oxidation by monocytes increased in a graded fashion as a function of stimulus concentration. Monocytes responded as a uniform population in response to increasing doses of PMA and HAIgG. This oxidative response was also monitored in mixed populations of mononuclear leukocytes, with monocytes identified on the basis of light scatter properties and surface antigen staining with anti-CD14. More than 90% of cells demonstrating increases in log green fluorescence signals following activation were CD14 positive. Measurement of DCFH oxidation by monocytes is reflective of the capacity to undergo a respiratory burst response, in that monocytes obtained from patients with chronic granulomatous disease were only minimally reactive. This assay, representing a rapid means of assessing monocyte respiratory burst activation by single cell analysis, is suitable for use in both clinical and research settings.