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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]

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]

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]