Phillip D. Smith

Isolation of whole mononuclear cells from peripheral blood and cord blood.

Peripheral blood is the primary source of lymphoid cells for investigation of the human immune system. Its use is facilitated by Ficoll‐Hypaque density gradient centrifugation—a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample. Thus, cells are distributed in the solution in layers based on the differences in their density/size. Additional purification methods can be employed as the mononuclear cell sample can be purified from monocytes by adherence or by exposure to L‐leucine methyl ester; these methods are described for both procedures. Cord blood and peripheral blood from infants contain immature cells, including nucleated red cells, which can result in significant contamination of the mononuclear cell layer, and removal of these cells requires additional steps that are described. The isolation procedures presented here can also be applied to cell populations derived from tissues. Curr. Protoc. Immunol. 85:7.1.1‐7.1.8.

Gastrointestinal Infections in AIDS

Abstract ▪ As the largest lymphoid organ in the body, the gastrointestinal tract is a potential reservoir for human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficienc...

Cytomegalovirus induction of tumor necrosis factor-alpha by human monocytes and mucosal macrophages.

Cytomegalovirus (CMV) is a major cause of inflammatory organ disease in immunosuppressed persons. To elucidate the mechanisms of CMV-induced inflammation, we investigated whether tumor necrosis factor-alpha (TNF-alpha) was involved in the pathogenesis of CMV colitis in patients with AIDS. In in situ hybridization experiments, TNF-alpha mRNA was shown to be abundantly present in colonic mucosa from AIDS patients with CMV colitis but not in colonic mucosa from control (AIDS and normal) subjects. The TNF-alpha transcripts, identified in macrophage-like cells containing cytomegalic inclusions, were positively associated with CMV, but not HIV-1, within the mucosa. In in vitro experiments, a patient-derived isolate of CMV, but not HIV-1Ba-L, induced human monocytes to express TNF-alpha mRNA and to release increased levels of TNF-alpha peptide following stimulation. CMV induction of TNF-alpha may play a critical role in CMV-induced inflammation and, since TNF-alpha upregulates expression of HIV-1, offers a mechanism by which CMV could serve as a co-factor for HIV-1 expression without both viruses infecting the same cell.

Endotoxin administration to humans primes alveolar macrophages for increased production of inflammatory mediators

To elucidate potential mechanisms of the acute lung injury associated with endotoxemia, we evaluated the effect of intravenously administered endotoxin on the ability of alveolar macrophages isolated by bronchoalveolar lavage from normal subjects to produce inflammatory mediators. Within 1 hr of endotoxin (4 ng/kg body weight) administration, all 12 study subjects developed constitutional symptoms and leukopenia, and within 3 hr, lowgrade fever. Resolution of symptoms and fever by 6 hr was accompanied by systemic granulocytosis. Although intravenously administered endotoxin appeared to activate a subset of circulating monocytes, it did not alter the bronchoalveolar lavage cell number, phenotype (95% macrophages), or constitutively expressed high levels of surface HLA-DR and O2−. In contrast, intravenous endotoxin primed the alveolar macrophages for enhanced lipopolysaccharide-induced secretion of interleukin-1 (11.8 to 25.8 U/ml;P=0.04), tumor necrosis factor-α (titer, 6.8 to 13.6;P=0.20), and prostaglandin E2 (38.4 to 116.3 ng/ml;P=0.035). These results demonstrate that low-dose intravenous endotoxin primes human alveolar macrophages, which are already differentiatedin situ, for enhanced secretion of inflammatory mediators. Such mediators may contribute to the pulmonary changes associated with endotoxemia and acute lung injury.

Human host response to Giardia lamblia: II. Antibody-dependent killing in vitro

We examined whether peripheral blood monocytes, lymphocytes, polymorphonuclear leukocytes, or eosinophils, obtained from individuals without known exposure to Giardia lamblia, exhibit antibody-dependent cellular cytotoxicity (ADCC) for this organism. Peripheral blood granulocytes but not lymphocytes from each individual were cytotoxic for G. lamblia in the presence of either human or rabbit anti-G. lamblia serum. At an effector to target ratio of 30:1, granulocyte cytotoxicity was 20% in the presence of human antiserum and 36% in the presence of rabbit antiserum. Granulocyte ADCC was concentration dependent with respect to antiserum and was not complement mediated. Most of the ADCC activity was mediated by polymorphonuclear neutrophils, and to a lesser extent by eosinophils. The predominant isotype of the antibodies sensitizing G. lamblia for granulocyte ADCC was IgG. We conclude that human peripheral blood granulocytes are cytotoxic for G. lamblia in the presence of IgG anti-G. lamblia antibodies. This activity, in addition to the previously observed spontaneous monocyte cytotoxicity, is likely to be an important mechanism for host defense against this parasite.

A phase I trial of recombinant human interferon-γ in patients with Kaposi's sarcoma and the acquired immunodeficiency syndrome (AIDS)

A Phase I study of recombinant interferon-gamma (rIFN-γ) was conducted to determine the toxicity and pharmacokinetics of this lymphokine in acquired immunodeficiency syndrome (AIDS) patients with Kaposis sarcoma (KS). Sixteen patients with AIDS/KS were entered into a fixed-dose trial at either 0.001, 0.01, 0.1, or 1.0 mg/m2 of rIFN-γ. rIFN-γ was initially administered either as a single 24-hr continuous iv infusion or as a single im injection, followed 4 days later by a 10-day course of daily therapy by the same route. Following a 1-week washout period, this sequence of administration was then repeated, with the drug given by the alternate route. Pharmacokinetic analysis of the 1.0-mg/m2 group revealed that peak serum levels of up to 153 U/ml occurred 2–4 hr after im injection and that steady-state levels of up to 40 U/ml were reached approximately 7–12 hr after beginning iv infusion. Dose-related toxicities in this trial included fever, headache, fatigue, nausea, and hepatitis, all of which were most severe at the two highest doses. Dose-dependent depression of the total white blood-cell (WBC) count, affecting both granulocytes and lymphocytes, was the most common laboratory abnormality. Natural killer (NK)-cell activity was slightly enhanced at a dose of 0.1 mg/m2 but suppressed at 1.0 mg/m2 of drug; monocyte-mediated cytotoxicity, in contrast, was significantly increased only at the highest dose. No dose-related changes were noted in KS lesions, HLA-DR expression by peripheral blood mononuclear cells, lymphocyte blastogenesis, or the ability to culture cytomegalovirus (CMV) from body fluids. We conclude that a maximally tolerated dose (MTD) for this drug is in the range of 0.1–1.0 mg/m2 and that at least modest evidence of systemic immunomodulation may be seen when rIFN-γ is given at doses at or near this MTD.

Isolation of Monocyte/Macrophage Populations

This unit describes the isolation of monocytes from lymphocytes by adherence, gradient sedimentation on colloidal silica particles, and flow cytometry. Because the first two methods can result in cell activation (induction of gene expression or protein secretion), and the third is technically difficult, a fourth protocol is presented which describes counterflow centrifugal elutriation. This latter procedure can be used to isolate large numbers of purified, nonactivated monocytes.

Defective spontaneous but normal antibody-dependent cytotoxicity for an extracellular protozoan parasite, Giardia lamblia, by C3H/HeJ mouse macrophages

To understand murine host responses to extracellular protozoa, the capacity of peritoneal macrophages to exhibit cytotoxicity for [3H]thymidine-labeled Giardia lamblia trophozoites was investigated. Resident peritoneal macrophages from C3H/HeN mice expressed spontaneous cytotoxicity for G. lamblia in a manner that was dependent on both time and effector cell number; this cytotoxic activity was increased with cells elicited by an intraperitoneal injection of thio-glycollate. In contrast, spontaneous cytotoxicity for G. lamblia by resident and thioglycollate-elicited peritoneal macrophages from C3H/HeJ mice was markedly reduced. In the presence of anti-G. lamblia serum (ADCC), however, peritoneal macrophages from both C3H/HeN and C3H/HeJ mice exhibited striking augmentation of their cytotoxic activity for G. lamblia to equivalent levels. We conclude that macrophages from C3H/HeJ mice express defective spontaneous cytotoxicity but normal ADCC for the extracellular protozoan parasite, G. lamblia. The dissociation between the expression of these two effector cell functions suggests that macrophage spontaneous cytotoxicity and ADCC for extracellular protozoa are mediated by separate macrophage functions.

Human Immune Responses to Giardia lamblia

Epidemiologic, clinical, and experimental observations indicate that man develops an immune response to Giardia lamblia. Studies of several giardiasis outbreaks (Moore et al., 1969; Barbour et al., 1976) show that individuals repeatedly exposed to G. lamblia have a lower incidence of infection and symptoms than newly exposed individuals, suggesting that prior exposure imparts partial resistance to reinfection. Clinical observations (Hughes et al., 1971; Ajdukiewicz et al., 1972; Ament et al., 1973; Brown et al., 1973; Hermans et al., 1976) of hypogammaglobulinemic patients reveal that these individuals have an increased prevalence of giardiasis, emphasizing the potential importance of antibody in the immunity to this parasite. In addition, a high prevalence of G. lamblia infection in homosexual men (Meyers et al., 1977; Hurwitz and Owen, 1978; Schmerin et al., 1978) suggests that cellular immunity may participate in man’s response to Giardia, since many homosexuals have an acquired T-cell defect (Masur et al., 1982; Siegal et al., 1982; Gottlieb et al., 1982). As discussed in Chapter 11, recent investigations (Roberts-Thomson et al., 1976; Stevens et al., 1978; Roberts-Thomson and Mitchell, 1978) in experimental G. muris infection in mice confirm the role of immunity in the murine response to Giardia, Recently, macrophages have also been implicated in immunity to this parasite, since macrophages of both mice (Owen et al., 1981) and rabbits (Radulescu and Meyer, 1981) are capable of phagocytosing Giardia.