Elderly persons have a high incidence of lethal infections by encapsulated bacteria. However, mechanisms involved in their poor defense and maintenance of immunological memory have been poorly understood. The present study characterized the population of B cells known as IgM memory B cell compartment and their response by pneumococcal vaccine in elderly people. CD27+ memory B cells, particularly IgD+IgM+CD27+ IgM memory B cells, had dramatically declined in the aged. Their Ig syntheses by B cells and the differentiation into plasma cells were diminished in vitro compared with those in adults. A rise of anti-pneumococcal IgM in sera of elderly persons was found with lower levels compared with those in adults after pneumococcal vaccination. Although diminished function itself of aged B cells surely exist, decline of the IgM memory B cell pool is expected to result in a poor humoral immunity against pneumococcal infection in elderly people.

SummaryHuman peripheral blood eosinophils released eosinophil survival-enhancing activity when stimulated with the calcium ionophore, ionomycin . The release of activity was detected as early as 3 h after stimulation and was inhibited by an immunomodulating agent, cyclosporin A . The survivalenhancing activity was completely abolished by treatment with anti-interleukin 3 (IL3) and anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) monoclonal antibodies . Moreover, IL-3 and GM-CSF were measurable in ionomycin-stimulated eosinophil supernatants by immunoassay. Eosinophils produced approximately one-half as much IL3 and one-fifth as much GM-CSF as ionomycin-stimulated mononuclear cells. Neutrophils also produced 11,3 and GM-CSF, but the amounts were less than those produced by eosinophils. These observations suggest a novel role for eosinophils in pathophysiology of allergic inflammation and host defense mechanisms. E osinophils are blood leukocytes associated with helminth infections and allergic diseases, especially bronchial asthma, and may mediate immunity and tissue damage (1) . Several rytokines, including IL3, 116, and granulocyte/macrophage colony-stimulating factor (GM-CSF), induce eosinophilopoiesis and activate mature peripheral blood eosinophils in vitro (reviewed in reference 2). Recently, mRNA for these rytokines was detected in allergen-induced late-phase cutaneous reactions in atopic patients, suggesting that these rytokines play an important role in allergic inflammation (3) .Because human peripheral blood eosinophils, despite their high degree of specialization, retain the ability to synthesize rytokines such as TGF-ot and IL1 (4, 5), we investigated whether they can produce IL-3, IL-5, and GM-CSF . We found that ionomycin-stimulated eosinophils produce GM-CSF and IL-3 as shown by enhanced eosinophil survival and by immunochemical measurement .Cells and Culture Conditions. Eosinophils, neutrophils, and mononuclear cells were isolated from peripheral blood of healthy volunteers or patients with mild hay fever by discontinuous Percoll density gradient as previously described (6) . Purities of eosinophils and neutrophils were 391% and 399%, respectively. Contaminating cells in the eosinophil preparations were only neutrophils and those in the neutrophil preparations were only eosinophils . Cell viabilities were >98% . Cells were incubated (2 x 105 cells per 0 .2 ml per well) in Hybri-Care medium (American Type Culture Collection, Rockville, MD) supplemented with 50 lAg/ml gentamicin and 10% defined calf serum in 96-well flat-bottomed microtiter plates (Falcon Labware, Lincoln Park, NJ) with ionomycin (Calbiochem Corp., San Diego, CA) in the presence or absence of PMA (500 pg/ml; Calbiochem Corp.) . After 24 h at 37°C, cellfree supernatants were harvested and frozen until assayed .Eosinophil Survival Assay. To measure eosinophil survival enhancing activity (7) in supernatants, eosinophils were freshly isolated from different donors and were cultured (2 .5 x 10" cells per 0 .2 ml per well) ...

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The effects of recombinant human (rh) interleukin (IL)-4 or rhIL-13 on survival, and chemotactic activity of human eosinophils were examined. Only rhIL-13 prolonged eosinophil survival in a dose-dependent manner above 3 ng/ml. Eosinophil survival induced by rhIL-13 was inhibited by monoclonal antibodies (mAbs) against IL-3 (p<0.0!) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (p<0.05), suggesting that rhIL-13 induced IL-3 and GM-CSF production from eosinophils and an autocrine mechanism is responsible for the eosinophil survival. The effects of rhIL-13 on eosinophil chemotactic activity were also examined. rhIL-13 showed chemotactic activity for eosinophils in a dose-dependent manner. Checkerboard analysis revealed that eosinophil migration was dependent on the concentration gradient, confirming that rhIL-13 is a chemotactic factor. rhIL-4 showed no effects. IL-13 may play an important role in the survival and recruitment of eosinophils in allergic diseases.

Neurological transmitters including ACh, substance P (SP), and calcitonin gene-related peptide (CGRP) play an important role in regulating airway tone, and increased bronchial reactivity to cholinergic stimulation is a well-recognized phenomenon in patients with bronchial asthma. We postulated that ACh, SP, and CGRP might stimulate alveolar macrophages (AMs) to release neutrophil, monocyte, and eosinophil chemotactic activities. To test this hypothesis, bovine AMs were isolated by bronchoalveolar lavage and cultured. AMs released chemotactic activities in response to ACh in a dose- and time-dependent manner ( P < 0.05). However, SP and CGRP did not stimulate bovine AMs. Checkerboard analysis revealed that these released activities were predominantly chemotactic. Partial characterization and molecular-sieve column chromatography revealed that low-molecular-weight lipid-soluble activity was predominant. Lipoxygenase inhibitors significantly blocked the release of chemotactic activities ( P < 0.05). Leukotriene B4- and platelet-activating factor-receptor antagonists blocked the chemotactic activities. Immunoreactive leukotriene B4 significantly increased in supernatant fluids in response to ACh ( P < 0.05), but platelet-activating factor did not. The receptor responsible for the release of the chemotactic activities was the muscarinic M3 receptor. These data demonstrate that ACh stimulates AMs to release lipoxygenase-derived chemotactic activities and plays a role in inflammatory cell recruitment into the airway.