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We have described functional and biochemical characteristics of a distinct T-cell dependent guinea- pig basophil-stimulating factor (BSF), measured using a sensitive 7-day bone marrow culture assay, standardized with high-activity BSF present in serum-free splenic cell-conditioned medium (CM). In the present studies, the in vivo relevance of BSF was explored during protocols of induction of peripheral blood or tissue basophil responses to ovalbumin (OA) injection or Amblyomma wnerkanwn tick feeding. Pooled immune serum, taken from OA-injected inbred or outbred animals during induction of blood and marrow basophilia, contained an in vitro inhibitor to BSF at high concentrations and BSF-like activity at low concentrations; maximal stimulation of histamine synthesis by bone marrow cells in vitro was found in the presence of Day 4 OA-immune serum. In vivo studies in the OA model demonstrated maximal serum BSF-like activity at 48-72 hr before peak bone marrow basophil response, followed by a levelling off to 50% of maximum at 2 weeks. In the tick model, serum BSF-like activity was present in Day 8, but not Day I, post-primary infection and was maximal at Day 3 post-secondary infection; post-primary Day I serum was inhibitory to basophil growth in vitro. These observations suggest that BSF regulates the appearance of basophils in response to antigen in vivo by an effect on basophil progenitors. The observations stress the potential application of guinea-pig models to understanding the regulation of basophil production in allergic disorders. [ABSTRACT FROM AUTHOR]

Intestinal mastocytosis begins to develop in rats, depending on the strain, at 14 (outbred Sprague-Dawley, SD) or 16 (inbred Lewis, L) days after infection with the nematode Nippostrongylus brasiliensis (Nippo). We have investigated in vitro mastopoiesis from mesenteric lymph node (MLN) cells cultured at various intervals post-infection, using a modified Marbrook liquid system. Greater increases in mast cells (MC) were observed in cultures of SD-MLN removed on day 14 after Nippo infection (IMLN-14) than from MLN removed from uninfected animals (NMLN): seven- to twenty-fold versus up to two-fold at 2 weeks and forty- to two hundred-fold versus up to twenty- fold at 4 weeks, respectively (P«0.002). In contrast, similar differential increases in MC and histamine compared to uninfected controls, were demonstrated in 2 week cultures of MLN from L strain rats removed 17 (IMLN-17) and 20 (IMLN-20) but not 14 days after Nippo infection (P«0.001). The presence of phytohaemagglutinin (PHA) in vitro was associated with enhanced MC differentiation from both IMLN-17 and IMLN-20, while worm antigen (Ag) stimulated mastopoiesis from IMLN-17, but suppressed the response from IMLN-20 (P « 0.02). Conditioned media (CM) prepared from unstimulated or PHA-stimulated IMLN-32 (i.e. removed 32 days after Nippo infection) caused significant mastopoiesis from NMLN ha vitro when compared to no CM or Ag-stimulated CM (P«0.01). Either MC precursors or cells which help MC differentiation exist in increased numbers in MLN of Nippo-infected rats. Mitogenic or antigenic stimulation modulates in vitro mastopoiesis, either directly or through soluble factors derivable from MLN cells. These in vitro methods can be utilized to understand further mechanisms of intestinal mastocytosis in the rat. [ABSTRACT FROM AUTHOR]

Hematopoiesis refers to the development of blood cells in the body through the differentiation of pluripotent stem cells. Although hematopoiesis is a multifocal process during embryonic development, under homeostatic conditions it occurs exclusively within the bone marrow. There, a limited number of hematopoietic stem cells differentiate into a rapidly proliferating population of lineage-restricted progenitors that serve to replenish circulating blood cells. However, emerging reports now suggest that under inflammatory conditions, alterations in hematopoiesis that occur outside of the bone marrow appear to constitute a conserved mechanism of innate immunity. Moreover, recent reports have identified previously unappreciated pathways that regulate the egress of hematopoietic progenitor cells from the bone marrow, alter their activation status, and skew their developmental potential. These studies suggest that progenitor cells contribute to inflammatory response by undergoing in situ hematopoiesis (ISH). In this review, we highlight the differences between homeostatic hematopoiesis, which occurs in the bone marrow, and ISH, which occurs at mucosal surfaces. Further, we highlight factors produced at local sites of inflammation that regulate hematopoietic progenitor cell responses and the development of TH2 cytokine-mediated inflammation. Finally, we discuss the therapeutic potential of targeting ISH in preventing the development of inflammation at mucosal sites. [ABSTRACT FROM AUTHOR]

We have examined the biochemical and functional characteristics of mast cells grown in tissue culture from the mesenteric lymph node (MLN) of rats infected with the nematode Nippostrongylus brasiliensis and compared them with mast cells isolated from the small intestinal mucosa and peritoneal cavity of infected animals. Cultured mast cells (MC) and isolated intestinal mucosal mast cells (MMC) had a similar histamine content, and both contained type II protease (RMCP II) which was absent from peritoneal mast cells (PMC). PMC, MMC and cultured MC each responded to immunologically induced histamine secretion, but MMC and cultured MC were hyporesponsive to calcium ionophores and unresponsive to widely used PMC secretagogues including compound 48/80 and bee venom peptide 401. MMC and cultured MC also differed from PMC in their lack of responsiveness to the anti-allergic agent disodium cromoglycate. Thus, MC cultured from the MLN are distinct from PMC but have a biochemical and functional phenotype similar to that of intestinal MMC. [ABSTRACT FROM AUTHOR]

Nasal polyposis is a chronic inflammatory condition of the upper airways characterized by infiltration of activated inflammatory cells, including mast cells, both in the epithelium and in the stroma. The aim of this work was to study human must cells derived from two different anatomical sites within the same nasal polyp tissue. To this end, we isolated two distinct mast cell populations, one from the epithelial and the other from the stromal layers of individual human nasal polyp tissues. We examined the mediator content of the two mast cell populations, and found that stromal mast cells had a significantly higher content of tryptase compared with the epithelial mast cells from the same tissue. In addition, mast cells from the stromal compartment, but not those from the epithelium, released a significant amount of histamine after anti-IgE stimulation. By contrast, both populations released over 50% of the total histamine after non-specific stimuli (A23187 10-6 M). The content of mediators and the response to immunological activation were not significantly altered in patients receiving topical steroid therapy. It remains to be determined if the observed differences are the result of an intrinsic characteristic of the mast cell populations localized to separate tissue compartments, or reflect a different in vivo exposure lo stimuli such as antigens, or different surrounding structural or infiltrating cells. In conclusion, these data provide evidence of functional heterogeneity and differences in mediator content between mast cell subpopulations from a single human tissue. The failure of release of epithelial mast cell mediators from an immunologic stimulus may have implications concerning acute effects of antigen exposure in nasal polyposis. [ABSTRACT FROM AUTHOR]

Recent candidate gene and genome-wide association studies have identified 'protective' associations between the single-nucleotide polymorphism (SNP) rs1837253 in the TSLP gene and risk for allergy, asthma, and airway hyperresponsiveness. The absence of linkage disequilibrium of rs1837253 with other SNPs in the region suggests it is likely a causal polymorphism for these associations, having functional consequences. We hypothesized that rs1837253 genotype would influence TSLP secretion from mucosal surfaces. We therefore evaluated the secretion of TSLP protein from primary nasal epithelial cells (NECs) of atopic and nonatopic individuals and its association with rs1837253 genotype. We found that although atopic sensitization does not affect the secretion of TSLP from NECs, there was decreased TSLP secretion in NECs obtained from heterozygous (CT; 1.8-fold) and homozygous minor allele (TT; 2.5-fold) individuals, as compared with NECs from homozygous major allele individuals (CC; P<0.05), after double-stranded RNA (dsRNA) stimulation (50 μg ml−1). Our novel results show that rs1837253 polymorphism may be directly involved in the regulation of TSLP secretion. This may help explain the protective association of this genetic variant with asthma and related traits. Identifying functional consequences of SNPs in genes with previously reported clinical associations is critical in understanding and targeting allergic inflammation. [ABSTRACT FROM AUTHOR]

Summary Eosinophils elaborate a number of proinflammatory mediators, including immunoregulatory cytokines and chemokines. Interleukin (IL)-4 and RANTES are important cytokines that have previously been shown to be expressed by mature eosinophils. We hypothesized that de novo synthesis of IL-4 and RANTES occurs in nascent eosinophils, leading to storage of newly produced proteins in crystalloid granule-like structures. Cytokine mRNA and protein expression were examined in cultured eosinophil colonies, which were derived from purified cord blood CD34+ cells and generated in semisolid media (methylcellulose) in the presence of recombinant human (rh)IL-3 and rhIL-5. Cytokine mRNA profiles were analysed by the reverse transcription–polymerase chain reaction (RT–PCR) to determine transcription of IL-4 and RANTES in cells on days 0, 7, 14, 21 and 28 of culture. The expression of translated cytokine products and granule major basic protein (MBP) was confirmed, from day 23 onwards, for colonies cultured in semisolid media, by immunofluorescent labelling and confocal laser-scanning microscopy (CLSM). We found that mRNA sequences encoding IL-4 and RANTES were expressed in freshly prepared, non-differentiated CD34+ cells. Furthermore, RANTES mRNA localized to carbol chromotrope 2R-positive colony cells, as assessed using in situ RT–PCR on day 21 of culture in semisolid media, and was found to gradually decrease (relative to β2-microglobulin) in rhIL-3- and rhIL-5-treated colony cells (comprising > 90% eosinophil-like cells) up to day 28. Immunoreactivity for IL-4 and RANTES co-localized with MBP in maturing colony eosinophils on day 23 of culture in semisolid media, as judged by CLSM. These results suggest that synthesis and storage of immunoregulatory cytokines, essential for processes associated with adaptive immunity, occurs in nascent eosinophils during their growth and differentiation. [ABSTRACT FROM AUTHOR]

The development of FcR∊-bearing histamine-containing basophilic cells was studied in cultures of peripheral blood leucocytes and bone marrow leucocytes from normal individuals. The determination of basophilic cells was performed blindly. Before cultivation there were fairly similar numbers of basophilic cells in the samples from the three different sources (1.6 ± 1.6 n= 13; 1.4 ± 2.0 n=21; 4.3 ± 4.7 n =8, respectively). During cultivation, spontaneously appearing large blast-like basophilic cells were seen in good correspondence with a formation of histamine in the cultures (Spearman rank correlation coefficient = 0.9135, n = 13. P < 0.001). This was more accentuated with bone marrow cells than with peripheral blood and cord blood cells. Conditioned medium (CM) was prepared from cells isolated from various tissues and stimulated by different means, e.g. peripheral blood from atopic individuals stimulated with allergen and unstimulated tonsil cells. Addition of the CM resulted in increased development of histamine-containing basophilic cells. Optimum stimulation was achieved with 10% CM. The basophilic stimulation by CM, as assessed as indices vs. unstimulated cultures, was more accentuated in cultures of peripheral blood cells than of bone marrow and cord blood cells (2.8 ± 1.2; 1.8 ± 0.5; 2.0 ± 0.4, respectively). In contrast, the histamine formation was particularly evident in stimulated cultures of bone marrow cells, where more than four-fold increases of histamine were found. In bone marrow cells the histamine levels per basophilic cell also increased, whereas this was not the case in cord blood cells. A pronounced development of basophilic cells was achieved when using leukoagglutinin, provided the mitogen in the CM was eliminated. The formation of basophilic cells was prevented with mitomycin c and cycloheximide. in conclusion, the system described may provide important information on the development of histamine-containing basophilic cells at various maturation stages from different compartments, and mechanisms in a developing atopic disease. [ABSTRACT FROM AUTHOR]