Increased numbers of circulating basophils with decreased releasability after administration of rhG-CSF to allergic patients

Preliminary studies in hematological patients have indicated that treatment with rhG-CSF reduces basophil releasabilityex vivo. We examined this phenomenon further, in allergic patients. Ten patients with grass pollen rhinoconjunctivitis were given rhG-CSF (5 g/kg/day s.c.) for 5 days, and examined before and after treatment. Basophil counts increased from 5 to 19×10⁹/l (P<0.01). total=" blood=" histamine=" increased=" from=" 80=" to=" 160=">g/l (P<0.01), corresponding=" to=" a=" decrease=" in=" average=" basophil=" histamine=" content=" from=" 1.5=" to=" 0.81=" pg/cell=">P<0.01). isolated=" mononuclear=" cells=" showed=" a=" significantly=" decreased=" histamine=" release=" (hr)=" when=" stimulated=" with=" a23187=" and=" grass.=" whole=" blood=" experiments=" showed=" a=" similar=" decreased=" hr=" to=" grass=" and=" anti-ige=">P<0.01). however,=" we=" found=" an=" increase=" in=" total=" blood=" histamine.=" we=" conclude=" that=" treatment=" with=" rhg-csf=" (1)=" increases=" the=" number=" of=" circulating=" blood=" basophils,=" (2)=" reduces=" the=" average=" histamine=" content=" per=" basophil,=" and=" (3)=" reduces=" the=" basophil=" releasability.=" these=" findings=" could=" be=" due=" to=" the=" mobilization=" of=" immature=" basophils=" from=" the=" bone=">     

Decreased releasability of basophils from patients with cold urticaria after cold exposure

Histamine release from peripheral blood basophils challenged with C5a, f-met-peptides and calcium ionophore was studied in patients with cold urticaria before and after exposure to low environmental temperatures. Compared to healthy controls, stimulated mediator release before cold exposure was increased in 7 of 11 patients. When challenged by cold exposure mediator release from in vitro-stimulated basophils was decreased. This decrease was more pronounced after stimulation with receptor-mediated stimuli (e.g. C5a) as compared to receptor-unrelated stimuli, e.g. calcium ionophore. In 4 of 11 patients stimulated mediator release before cold exposure was moderately increased. Also after cold exposure only a weak decrease of stimulated histamine release was seen. Levels of activated complement components (C3a) before and after cold exposure failed to provide evidence for complement activation in vivo. Also the number of circulating basophils as well as their cellular histamine content remained normal after cold exposure. The results show that in these patients release of histamine is altered before and after cold exposure. These changes in basophil responsiveness are not due to complement activation in vivo.     

Increased releasability of skin mast cells after exercise in patients with exercise-induced asthma

The role of lung mast cells in exercise-induced asthma (EIA) is controversial. To investigate whether the skin mast cell releasability is increased after exercise in EIA, 49 young atopic men with or without asthma took part in a free-running test for 6 min and were given skin prick tests using morphine, a mast cell secretagogue, before and after the exercise. The mean diameters of the wheal induced by morphine in patients with EIA were not significantly different from those in patients without EIA before exercise, although the baseline lung function was significantly lower and the airway hyperresponsiveness, the peripheral blood eosinophil count, and the size of the wheal in response to Dermatophagoides pteronyssinus were significantly higher in patients with EIA. However, the differences of the morphine-induced wheal diameter between patients with EIA and those without EIA became significant at 120 min after exercise (p<0.05), while the responses to histamine were not significantly different. These results suggest that exercise increases the releasability of skin mast cells in EIA patients whose asthma/allergy are relatively severe.     

Increased numbers of circulating basophil progenitors in atopic patients

Recruitment of basophils to sites of homocytotropic antibody-mediated hypersensitivity reactions has been well documented in both experimental and clinical situations. Mechanisms underlying tissue basophil accumulation, however, remain unclear and may involve chemotaxis, cell proliferation, or both. We have recently reported the presence in human blood of circulating basophil/mast cell progenitors on the basis of histamine content of granulocyte colonies grown in methylcellulose. In the current studies we have analyzed the peripheral blood of 30 patients with atopy and 25 comparable control subjects for frequency of basophil/mast cell progenitors by analysis of the histamine content of individual granulocyte colonies. Forty percent of granulocyte colonies in cultures of atopic patients contained histamine in comparison to only 11% in cultures of control subjects (p less than 0.001). Histamine content per colony as well as mean histamine per cell in each colony was higher in granulocyte colonies of atopic subjects and could not be related to colony size or culture conditions. Granulocyte colony growth was enhanced by antigen-stimulated, peripheral blood lymphomononuclear cell--conditioned media of atopic patients. Histamine-positive colonies were found more frequently in active versus quiescent atopic disease (p less than 0.05). These results are consistent with the hypothesis that basophils accumulate at sites of allergic reactions at least in part by recruitment of progenitors from circulation and subsequent differentiation in situ in response to lymphokines. Further studies by use of hemopoietic assays could elucidate the contribution of basophil production to the development of allergic conditions.     

Increased numbers of circulating donor-specific T helper lymphocytes after human heart valve transplantation

Implantation of cryopreserved human donor heart valves for either congenital or acquired cardiac disease has been performed since the last three decades. Although the clinical outcome is good, long-term valve degeneration resulting in dysfunction has been observed. A specific immunological response of the recipient against the allograft has been proposed as one of the factors involved in this process. Helper T lymphocytes play an important intermediate role in cellular and humoral immune response. Increasing numbers of circulating donor-specific helper T lymphocytes precursors (HTLp) correlate with graft rejection after organ transplantation. To investigate whether cryopreserved human donor heart valves are able to induce a donor-specific T helper response, we monitored the HTLp frequencies (HTLpf) in peripheral blood samples of 13 patients after valve allograft transplantation by use of a limiting dilution assay followed by an interleukin-2 bioassay. Prior to transplantation, HTLpf specific for donor and third-party antigens showed individual baseline levels. After allografting, the antidonor frequencies significantly increased in 11 of the 13 patients (P = 0.02). This was not found for stimulation with third-party spleen cells (P = 0.68), which indicates a donor-specific response. Maximal donor-specific HTLpf were already found at 1--2 months after operation. Valve allograft transplantation induces an increase in the numbers of donor-specific HTLp in peripheral blood of the patients. Analogous to organ transplantation, these HTLp may play a crucial role in events that lead to valve damage. Therefore, monitoring of HTLp in peripheral blood samples might be informative for donor valve degeneration (rejection) and subsequently valve allograft failure.     

Two populations of human blood basophils: effect of prednisone on circulating numbers

In a previous study, human blood basophils were separated by centrifugation on Percoll gradients into two fractions, band 1 and band 2, that differed in density and histamine content. In this study, the change in circulating numbers of band 1 and band 2 basophils, as well as other leukocytes, was measured after a single oral dose of prednisone. Three hours after ingestion of 50 mg of prednisone, the circulating number of band 1 basophils was 19 +/- 4% of the preprednisone value, whereas the band 2 level was 87 +/- 18% (SEM for five subjects). At 6 hours, values were 10 +/- 1% and 41 +/- 7% for band 1 and band 2, respectively. Circulating numbers of both basophil populations returned to near normal at 24 hours. The 3-hour response to a 10 mg oral dose in seven subjects was 28 +/- 5% and 89 +/- 10% of preprednisone levels for band 1 and band 2; the 6-hour responses were 28 +/- 7% and 84 +/- 7%. The 3-hour responses of other leukocytes in four of these subjects, expressed as a percentage of preprednisone counts were neutrophils, 171 +/- 27%; lymphocytes, 47 +/- 6%; monocytes, 36 +/- 9%; and eosinophils, 26 +/- 11%. The results demonstrate that band 2 basophils have a lower sensitivity to glucocorticoid action than do band 1 basophils or other types of circulating leukocytes.     

Increased responsiveness of basophils of patients with chronic urticaria to sera but hypo-responsiveness to other stimuli

BACKGROUND: Although it has been shown that basophils from patients with chronic ordinary urticaria (CU) are less responsive than normal basophils when stimulated with anti-IgE, very few studies have examined the response of those cells to alternative stimuli. OBJECTIVE: To compare releasability between basophils from healthy donors and patients with CU. METHODS: We examined the response of IL-3-treated basophils from healthy donors, atopic controls and CU patients to anti-IgE, monocyte chemoattractant protein-1 (MCP-1), bradykinin, C5a and to sera obtained from other urticaria patients and normal controls. We also compared the response of basophils from CU patients whose sera activate normal basophils (autoimmune CU) from those who do not (idiopathic CU). RESULTS: Basophils of CU patients release significantly less histamine than basophils of normal controls when stimulated with anti-IgE, and to a lesser degree with C5a. No differences were observed when basophils from patients were incubated with Bradykinin or MCP-1. However, when basophils of CU patients were incubated with sera from other CU patients or even normal sera, we found significantly higher histamine release compared with the response of basophils from normal donors. We could not distinguish responsiveness of basophils of patients with chronic autoimmune urticaria from patients with chronic idiopathic urticaria. CONCLUSION: Basophils of patients with chronic idiopathic urticaria and chronic autoimmune urticaria are hypo-responsive to anti-IgE and C5a, normally responsive to MCP-1 or bradykinin, and hyper-responsive to serum. The serum factor to which a response has not yet been identified; however, basophils of patients with chronic urticaria, in general, appear to have abnormal regulation of signaling pathways.     

Increased sensitivity to IL-4 in patients with allergic bronchopulmonary aspergillosis

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by a heightened Th2 CD4+ cell response to Aspergillus fumigatus allergens and a hyper-IgE state compared to atopic asthmatic and cystic fibrosis patients without ABPA. We hypothesized that one reason for this response is increased sensitivity to IL-4 in ABPA, resulting in increased expression of CD23 and CD86, leading to a positive amplification mechanism which increases Th2 CD4+ T cell responses. METHODS: Peripheral blood mononuclear cells isolated from 10 ABPA, 9 atopic, and 8 nonatopic subjects and stimulated for 48 h with varying concentrations of rIL-4 ranging from 0.1 to 50 ng/ml. The percentages of CD23+ and CD86+ B cells and the number of CD23+ molecules on CD20+ and CD86+CD20+ B cells were quantified by flow cytometry. RESULTS: Total serum IgE levels were elevated in ABPA patients compared to atopic and nonatopic controls. At day 0 prior to culture, CD23 molecules per CD20+ B cell were significantly elevated in ABPA patients compared to atopic and to nonatopic patients. CD23 molecules per CD20+ B cell in ABPA and atopic patients decreased after 48 h in culture without IL-4 added and were similar. With IL-4 stimulation, ABPA patients had significantly increased rates of CD23 expression per B cell compared to atopic and nonatopic subjects (p < 0.001). Furthermore, ABPA had significantly increased numbers of CD23+ molecules per B cell and CD86+ B cell following IL-4 stimulation compared to atopic and nonatopic patients. Both ABPA and atopic patients at day 0 prior to culture had increased expression of CD86+ and CD23+CD86+ B cells compared to nonatopic patients. After 48 h in culture without IL-4, the percentages of CD86+ and CD23+CD86+ B cells decreased in ABPA and atopic patients. After stimulation with IL-4, ABPA patients had significant upregulation of CD23+CD86+ B cells compared to atopic and nonatopic patients. Similarly, the number of CD23 molecules per CD86+CD20+ B cell was significantly upregulated following IL-4 stimulation in ABPA patients compared to atopic and to nonatopic subjects. CONCLUSIONS: This is the first study to demonstrate that ABPA patients have increased sensitivity to IL-4 stimulation compared to other atopic individuals, such that ABPA > atopic > nonatopic patients. The B cells from ABPA patients were significantly more sensitive to IL-4 stimulation compared to atopic and nonatopic patients with upregulation of CD23 and CD86 expression. ABPA subjects had increased CD86+ and CD23+CD86+ B cell expression on day 0 prior to culture and with upregulation of CD23+ molecules on CD86+CD20+ B cells. IL-4 also stimulated upregulated CD86+ expression on B cells in atopic patients with little effect on nonatopic patients. This study supports the premise that IL-4, IL-4R alpha and CD86 are central targets in the treatment of ABPA and atopic disease.     

Differential mediator release from basophils of allergic and non-allergic asthmatic patients after stimulation with anti-IgE and C5a

The differentiation between allergic and non-allergic asthma is a common and important challenge for the clinician. Until now, no in vitro diagnostic characteristics have been described to distinguish between these types. To examine the diagnostic value of a basophil stimulation test, we compared anti-IgE- and C5a-induced mediator release from peripheral blood leucocytes in different types of bronchial asthma. Peripheral blood leucocytes (PBL) from 10 aspirin-sensitive asthmatics (ASA), 12 non-allergic asthmatics without aspirin intolerance (NAA), seven allergic asthmatics (AA), and nine healthy controls were prepared by dextran sedimentation. After priming with interleukin-3 (IL-3) PBL were stimulated with anti-IgE and C5a, and the release of histamine (HR) and sulfidoleukotrienes (LTR) in the supernatant was compared. Additionally, purified leucocyte fractions were studied to determine the cellular source of mediator release. Upon stimulation with anti-IgE LTR was slightly, but not significantly, lower in ASA and NAA compared to AA and controls. In contrast, C5a-triggered LTR was significantly higher in ASA (14.4 +/- 12.88 pg/105 cells) and NAA (22.9 +/- 22.61 pg/105 cells) than in AA (9.6 +/- 3.29 pg/105 cells) and controls (7.5 +/- 7.19 pg/105 cells) (P < 0.05). This difference between ASA and NAA vs. AA and controls was even more pronounced when determining the quotient C5a-/anti-IgE-induced LTR (P < 0.001). At an optimal cut-off point of 1.0, calculated by relative operating characteristics (ROC) analysis, the positive predictive value for a donor to belong to ASA or NAA was 0.94. No significant differences could be found in HR between the asthmatic patient groups and healthy controls in either condition. As cellular source of LTR and HR the basophil could be determined. Determination of anti-IgE- and C5a-induced LTR from basophils allows us to discriminate between allergic and non-allergic asthmatic patients. For diagnostic purposes the quotient C5a-/anti-IgE-induced LTR is more significant than considering a single parameter. ASA cannot be distinguished from NAA.     

Increased circulating CCR3+ type 2 helper T cells in house dust mite-sensitive Chinese patients with allergic diseases

Chemokine receptor expression has been shown to be associated with the differentiation of T helper cells. The CCR3, CXCR4 and CCR5 expression on circulating T cells were studied in 30 house dust mite sensitive-patients with allergic diseases and in another 30 healthy controls. The expression was analyzed in CD4, CD8 and double negative (DN) T cells by triple fluorescence staining. In addition, intracellular cytokine staining was performed in the CCR3+ CD4+ T cells. Increased circulating portions of CCR3+ CD4+ T cells and CCR3+ DN T cells were found in these patients (p < 0.01). There was no statistically significant difference in the expression of CXCR4 and CCR5 on T cells. The follow-up data of the patients did not show a statistically significant change in the CCR3 expression. IL-4 was expressed within CCR3+ CD4+ T cells upon activation. The IL-4 secreting CCR3+ type 2 T helper cells may play a pathogenetic role in immune responses of house dust mite-sensitive Chinese patients with allergic diseases.     

Increased phagocytic capacity of the blood,but decreased phagocytic activity per individual circulating neutrophil after an ultradistance run

The effect of a long strenuous endurance exercise on the phagocytic function of neutrophils was examined. 9 athletes [7 males, 2 females, age: 36–68 years, body mass: 64 (SD 10) kg, height: 175 (SD 10) cm] completed a competetive 100 km run in 8:07 (median value; range: 7:29–9:50 hours). In a whole blood assay the phagocytosis of opsonized E. coli, the receptor density of the Fc receptor 3 (CD16) and the complement receptor 3 (CD11b, direct immunofluorescence) of neutrophils were measured on a per cell basis by flow cytometry before and up to 3 hours after the race. The phagocytic rate (percentage of neutrophils incorporating bacteria) was unchanged after exercise, whereas the phagocytic activity (number of incorporated bacteria per cell) was significantly reduced by –34 (SD 8) % (Wilcoxon test, P<0.001). The total phagocytic capacity of the blood increased 2-3fold post exercise. The surface antigen expressions of CD11b and CD16 were unaffected by the ultradistance run. The results indicate either a reduced phagocytic function of neutrophils on a single cell basis or the mobilization of neutrophils of the marginal pool with a lower phagocytic activity. However, after a long endurance exercise the phagocytotic capacity of the blood was enhanced due to increased cell concentrations.     

Increased sensitivity to IL-4 in cystic fibrosis patients with allergic bronchopulmonary aspergillosis

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by a heightened Th2 CD4+ T-cell response to Aspergillus fumigatus allergens and a hyper-immunoglobulin (Ig)E state compared with cystic fibrosis patients without ABPA. We hypothesize that one reason for this response is increased sensitivity to interleukin (IL)-4 in ABPA resulting in increased expression of CD23 and CD86 and leading to a positive amplification mechanism that increases Th2 CD4+ T cell responses. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from seven ABPA CF and 19 non-ABPA CF patients and 16 nonatopic controls and stimulated with rIL-4 (range 0.1-10 ng/ml) and rIL-13 (range 1-10 ng/ml) for 48 h. The number of CD23 molecules and percentages of CD23+ B cells were quantified by flow cytometry. Both phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO) and antigen stimulated, toxoid and Asp f2/f3/f4, PBMC were examined for cytoplasmic cytokine synthesis enumerated by cytokine staining using flow cytometry to measure Th2 and Th1 CD3+ T cells. RESULTS: The numbers of CD23 molecules on B-cells were significantly elevated at time 0 in ABPA CF patients compared with both non-ABPA CF patients and nonatopic controls. Following IL-4 stimulation in vitro, the numbers and percentages of CD23 expression on B cells were significantly up-regulated in ABPA CF patients compared with non-ABPA CF patients and controls. The IL-13 stimulation up-regulated CD23 expression; however, there was no significant difference in ABPA CF patients compared with non-ABPA CF patients and controls. The percentages of interferon (IFN)-gamma+ CD3+T cells following PMA/IO stimulation were significantly decreased in both ABPA and non-ABPA CF patients compared with controls. There were no significant differences of IL-4+ and IL-13+ CD3+ T cells between ABPA and non-ABPA CF patients. When tetanus toxoid stimulated T cells were examined, both ABPA and non-ABPA CF patients had significantly decreased IFN-gamma+ CD3+ T cells compared with controls. In Asp f2/f3/f4 stimulated T cells, ABPA CF patients had significantly increased IL-4+ CD3+ T cells compared with non-ABPA CF patients and controls. CONCLUSIONS: ABPA CF patients have increased sensitivity to IL-4 but not to IL-13 up-regulation of CD23 molecules compared with non-ABPA CF patients. There were decreased percentages of IFN-gamma+ and IL-2+ Th1 T cells in CF patients compared with nonatopic controls but similar percentages of IL-4+ Th2 T cells in all three groups. However, ABPA CF patients had increased frequency of Aspergillus-stimulated Th2 T cells. This indicated that there is skewing of Th2 T cells in ABPA CF patients. Thus, in CF ABPA patients there is increased Th2 T cells and increased sensitivity to IL-4.     

Increased sensitivity to cockroaches of the species Blattella germanica in patients with allergic diseases

Sensitization to Blattella germanica (B.g.) was studied in 290 allergic patients having increased sensitivity to house dust (HD). The study carried out by ELISA techniques with the use of allergen especially developed by the authors, revealed that the sera of adults and children with bronchial asthma (BA) contained high levels of IgE antibodies (Ab) to B.g. in 34 and 63.5% of cases, respectively. The presence of pronounced IgE-linked sensitization of target cells of BA patients was confirmed in leukocyte alteration test and in the natural leukocyte migration inhibition test. Increased sensitization to B.g. was shown to be the second in the total spectrum of sensitization to different arthropod species in house dust, and the presence of mixed sensitization to B.g. and HD mites was not due to cross reactions caused by main mite allergens. The content of IgG and IgG4 Ab to B.g. in BA patients exceeds that in healthy subjects, the tendency towards reverse correlation (r = -0.4) between the content of specific IgE and IgG Ab being revealed in the former. An essential role of sensitization to cockroaches in pathogenesis of BA is emphasized.     

Association of the Src homology 2 domain-containing inositol 5' phosphatase (SHIP) to releasability in human basophils

During the study of the biology of the Human recombinant Histamine Releasing Factor (HrHRF), we uncovered a hyperreleasable phenotype of basophils from HrHRF-responder donors. Basophils from these donors released histamne to HrHRF, IL-3 and D(2)O. While there has been a significant amount of work elucidating signal transduction events in human basophils, the reason for this hyperreleasable phenotype remained illusive. A clue to the releasability of these highly allergic, asthmatic HrHRF-responder donor basophils was demonstrated in studies using SHIP knockout mice. Bone marrow-derived mast cells from the SHIP knockout mice demonstrated hyperreleasability to stimuli through the IgE receptor and alteration of subsequent signal transduction events. We have demonstrated a highly significant negative correlation between the amount of SHIP protein per cell equivalent and maximum histamine release to HrHRF. These results provide a clue to the hyperreleasable phenotype and implicate SHIP as an additional regulator of secretion in human basophils.     

Failure to detect circulating immune complexes in allergic patients on injection therapy

Injection therapy for allergic diseases may create an environment conducive to circulating immune complex formation. Therefore, we examined the sera of eleven patients receiving injection therapy for the presence of circulating immune complexes. A sensitive Raji cell radioimmune assay was used to examine the sera prior to and 4, 8 and 24 hours after allergenic injection. Levels measured in these sera were compared to values obtained from forty-two healthy controls. Ten of eleven allergic patients receiving injection therapy had values within the normal range prior to maintenance injection. These ten patients also had normal values for circulating immune complexes at each interval after maintenance injection. The data suggest that circulating immune complexes are not a routine consequence of injection therapy for allergic disease.     

Histamine release induced by anti-IgE serum on basophils of normal and allergic patients

Conclusion These experimental conditions allow us to show the difference in the percentage of net histamine release induced by anti-IgE: weak in healthy subjects (10%) and strongly increased in allergic patients or patients with drug hypersensitivity (mean 35%).Grant of INSERM (74.1.024.3.).     

Increased levels of circulating interleukin 6 in burn patients

The serum levels of interleukin 6 (IL-6) were determined in a population of burn patients. In all patients, IL-6 levels were increased over a 3-week interval with peak concentrations reached during the first week after injury. Patients receiving intravenous polymyxin B therapy according to a regimen designed to reduce endotoxemia manifested greatly reduced levels of both circulating endotoxins and IL-6. Certain patients not treated with polymyxin B showed extraordinarily large increases in IL-6 which were associated with lethal or life-threatening clinical complications. Increased IL-6 levels were also associated with decreased percentage of circulating T cells and corresponding increases in B cells. However, IL-6 did not produce any direct inhibitory effects in vitro on T cell representation or function.