Review article

Detection of human cytokines in situ using antibody and probe based methods

Pancytopenia, hepatosplenomegaly and skeletal complications are hallmarks of Gaucher disease. Monitoring of the outcome of therapy on skeletal status of Gaucher patients is problematic since currently available imaging techniques are expensive and not widely accessible. The availability of a blood test that relates to skeletal manifestations would be very valuable. We here report that macrophage inflammatory protein (MIP)-1α and MIP-1β, both implicated in skeletal complications in multiple myeloma (MM), are significantly elevated in plasma of Gaucher patients. Plasma MIP-1α of patients (median 78 pg/ml, range 21–550 pg/ml, n = 48) is elevated (normal median 9 pg/ml, range 0–208 pg/ml, n = 39). Plasma MIP-1β of patients (median 201 pg/ml, range 59–647 pg/ml, n = 49) is even more pronouncedly increased (normal median 17 pg/ml, range 1–41 pg/ml, n = 39; one outlier: 122 pg/ml). The increase in plasma MIP-1β levels of Gaucher patients is associated with skeletal disease. The plasma levels of both chemokines decrease upon effective therapy. Lack of reduction of plasma MIP-1β below 85 pg/ml during 5 years of therapy was observed in patients with ongoing skeletal disease. In conclusion, MIP-1α and MIP-1β are elevated in plasma of Gaucher patients and remaining high levels of MIP-1β during therapy seem associated with ongoing skeletal disease.

The assessment of cytokine production is an important component of studies of cell-mediated immune responses (CMI) to immunological challenges. In this study, we present a method to enhance the detection of cytokine-producing cells by allowing antigen-specific cells to expand in long-term culture. We investigated the influence of the degree of dilution of whole blood and the duration of the incubation period on whole blood as well as peripheral blood mononuclear cells (PBMCs), cultured in the absence or presence of mitogens, superantigens or specific antigens, for intracellular cytokine production (IFNγ, TNFα, IL-2, IL-4, IL-10 and IL-13) by CD4⁺ and CD8⁺ T lymphocytes using four-colour flow cytometry. Whole blood was diluted 1/1, 1/2, 1/5 and 1/10, and cultured for 6, 24, 48, 72 and 120 h in the presence of antibodies against the co-stimulatory molecules CD28 and CD49d, and, during the last 4 h of culture, in the presence of brefeldin A. Optimum conditions for detection of a high number of IFNγ-positive cells were observed after 72 h of culture in blood diluted 1/10. Median frequencies of IFNγ⁺ cells obtained after activation by PMA-ionomycin, PHA or SEA-B were 29.3%, 20.0% and 6.8% for CD4⁺ cells, and 67.8%, 20.6% and 6.8% for CD8⁺ cells. In blood samples diluted 1/5 or 1/10, and cultured in the presence of cytomegalovirus (CMV) or varicella-zoster virus (VZV), mean peak levels of 2.8% and 1.4% IFNγ⁺CD4⁺ cells were recorded at 120 h. The levels of cells producing cytokines other than IFNγ were generally much lower and, in the case of IL-4 and IL-13, difficult to distinguish from background levels recorded in cultures with medium only. Kinetic studies of cytokine production by PBMCs showed a pattern similar to that of whole blood with peak levels of IFNγ-producing cells recorded at 72 h. The increased levels of IFNγ production after culture for 72 h were due to an expansion of the numbers of cytokine-producing cells responsive to a specific stimulus. Antigen-specific cells are usually present only at low levels in peripheral blood and may not be detected following simple activation for a few hours. To reach a level of detection in such cases, culture of diluted blood for several days is recommended.

Gaucher disease is characterized by storage of glucosylceramide in lysosomes of tissue macrophages as the result of an autosomal recessively inherited deficiency in glucocerebrosidase. Progressive accumulation of these glycolipid-laden Gaucher cells causes a variety of debilitating symptoms. The disease can be effectively treated by costly intravenous infusions with recombinant glucocerebrosidase. Chitotriosidase is massively secreted by Gaucher cells and its plasma levels are used to monitor efficacy of enzyme therapy. Broad-scale application is hampered by the common genetic defect in this surrogate marker. We report that in plasma of symptomatic patients with Gaucher disease the chemokine CCL18 is on average 29-fold elevated, without overlap between patient and control values (median control plasma level is 33 ng/mL, range, 10-72 ng/mL; median Gaucher plasma level is 948 ng/mL, range, 237-2285 ng/mL). Plasma CCL18 concentrations decrease during therapy, comparably to chitotriosidase levels. Immunohistochemistry demonstrates that Gaucher cells are the prominent source of CCL18. Plasma CCL18 levels can serve as alternative surrogate marker for storage cells in patients with Gaucher disease and monitoring of plasma CCL18 levels proves to be useful in determination of therapeutic efficacy, especially in patients who are deficient in chitotriosidase activity. The potential physiologic consequences of chronically elevated CCL18 in patients with Gaucher disease are discussed.

In a longitudinal prospective study a muscle biopsy was taken from 30/32 (33%) of the 98 patients who developed critical illness polyneuropathy and myopathy (CIPNM). Neuropathic changes were found in 37%, myopathic in 40%, and a combination in 23% of the biopsies. The immunohistopathology showed macrophages and Th-cells in 40% and 60% of the muscle biopsies respectively. Small mainly perivascular infiltrates contained macrophages and Th-cells. ICAM-1, VCAM and MAC were found on the vascular endothelium in 58%, 53% and 79% respectively. In all biopsies there was an upregulation of both HLA-I and HLA-DR. Proinflammatory cytokines and TNFαR75 were also produced locally (IL-1β in 71%, IFN-γ in 40%, IL-12 in 73%, TNFαR75 in 90%). The anti-inflammatory cytokine IL-10 was simultaneously expressed in 96% of the biopsies. HLA-DR, TNFαR75 and IL-10 differed significantly when compared with control muscle biopsies. Our data provide evidence that small numbers of activated leukocytes producing both pro- and anti-inflammatory cytokines infiltrate skeletal muscle of CIPNM patients. We propose that the local balance of leukocyte activities is of importance in the pathophysiology of muscle weakness in CIPNM.

The pathogenesis of Langerhans cell histiocytosis (LCH) is obscure, partly because the events leading to activation of Langerhans-like lesional cells (LCH cells) and associated T cells, and the excessive cytokine production by these cells are unknown. The interaction between CD40 on antigen-presenting cells (APC) like Langerhans cells and CD40 ligand (CD40L) (CD154) expressed by activated CD4+ T cells, is essential for the activation of both the APC and the T cells and results in upregulation of APC functions and initiation of immunoreactivity. The effects of CD40–CD40L interaction include increased expression of co-stimulatory and adhesion molecules, proliferation, and production of pro-inflammatory cytokines and proteolytic enzymes, all features of LCH. Using immunohistochemistry, we analysed the in situ presence of the co-stimulatory molecules CD40 and CD40L in 15 fresh frozen biopsies of LCH lesions in children. The cells producing these molecules were identified by double staining for CD1a on LCH cells and CD3 on T cells. Prominent expression of CD40 by LCH cells and CD40L by T cells was found in all 15 specimens regardless of the source of specimen or characteristics of the patient. The findings of high expression of CD40 and CD40L in all specimens imply a key role for the CD40–CD40L adhesion pathway in the pathogenesis of LCH. Since this interaction is an accessible and realistic target for immunotherapy, these findings prompt speculation on the use of blocking antibodies to CD40 or to CD40L in the treatment of LCH.

Erythema migrans, the characteristic skin manifestation of acute Lyme borreliosis, is a self-limited lesion. In contrast, acrodermatitis chronica atrophicans, the typical cutaneous manifestation of late Lyme borreliosis, is a chronic skin condition. In an effort to understand pathogenic factors that lead to different outcomes in dermatoborrelioses, skin biopsy samples from 42 patients with erythema migrans and 27 patients with acrodermatitis chronica atrophicans were analyzed for mRNA expression of five pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β, interleukin-6, interferon-γ, and interleukin-2) and two anti-inflammatory cytokines (interleukin-4 and interleukin-10) by in situ hybridization with cytokine-specific riboprobes. Among the 27 patients who had erythema migrans alone with no associated signs or symptoms, the major cytokines expressed in perivascular infiltrates of T cells and macrophages were the pro-inflammatory cytokine interferon-γ and the anti-inflammatory cytokine interleukin-10. In the 15 erythema migrans patients who had associated signs and symptoms, including headache, elevated temperature, arthralgias, myalgias, or fatigue, a larger number of macrophages and greater expression of macrophage-derived pro-inflammatory cytokines, tumor necrosis factor α, interleukin-1β, and interleukin-6, were also found. In comparison, infiltrates of T cells and macrophages in the skin lesions of acrodermatitis chronica atrophicans patients had very little or no interferon-γ expression. Instead, they usually expressed only the pro-inflammatory cytokine tumor necrosis factor α and the anti-inflammatory cytokine interleukin-4. Thus, the activation of pro-inflammatory cytokines in erythema migrans lesions, particularly interferon-γ, seems to be important in the control of the spirochetal infection. In contrast, the restricted pattern of cytokine expression in acrodermatitis chronica atrophicans, including the lack of interferon-γ, may be less effective in spirochetal killing, resulting in the chronicity of this skin lesion.