The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (MØ) are commonly drawn for functional studies. Here we define two MØ subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal MØ (LPM), contains approximately 90% of the PerC MØ in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical MØ surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal MØ (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of MØ heterogeneity and shed new light on PerC MØ diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC MØ.CD11b | F4/80 | lipopolysaccharide | peritoneal cavity | thioglycolate

Glutathione (GSH) deficiency is associated with numerous pathological conditions. Administration of N-acetylcysteine (NAC), a cysteine prodrug, replenishes intracellular GSH levels. NAC, best known for its ability to counter acetaminophen toxicity, is a safe, well-tolerated antidote for cysteine/GSH deficiency. NAC has been used successfully to treat GSH deficiency in a wide range of infections, genetic defects and metabolic disorders, including HIV infection and COPD. Over two-thirds of 46 placebo-controlled clinical trials with orally administered NAC have indicated beneficial effects of NAC measured either as trial endpoints or as general measures of improvement in quality of life and well-being of the patients.

We have studied the role of secreted immunoglobulin (Ig)M in protection from infection with influenza virus and delineated the relative contributions of B-1 versus B-2 cell–derived IgM in this process. Mice deficient in secreted IgM but capable of expressing surface IgM and secreting other Ig classes show significantly reduced virus clearance and survival rates compared with wild-type controls. Irradiation chimeras in which only either B-1 or B-2 cells lack the ability to secrete IgM show mortality rates similar to those of mice in which neither B-1 nor B-2 cells secrete IgM. Dependence on both sources of IgM for survival is partially explained by findings in allotype chimeras that broadly cross-reactive B-1 cell–derived natural IgM is present before infection, whereas virus strain–specific, B-2 cell–derived IgM appears only after infection. Furthermore, lack of IgM secreted from one or both sources significantly impairs the antiviral IgG response. Reconstitution of chimeras lacking B-1 cell–derived IgM only with IgM-containing serum from noninfected mice improved both survival rates and serum levels of virus-specific IgG. Thus, virus-induced IgM must be secreted in the presence of natural IgM for efficient induction of specific IgG and for immune protection, identifying B-1 and B-2 cell–derived IgM antibodies as nonredundant components of the antiviral response.

Natural antibodies are produced at tightly regulated levels in the complete absence of external antigenic stimulation. They provide immediate, early and broad protection against pathogens, making them a crucial non-redundant component of the humoral immune system. These antibodies are produced mainly, if not exclusively, by a subset of long-lived, self-replenishing B cells termed B-1 cells. We argue here that the unique developmental pattern of these B-1 cells, which rests on positive selection by self antigens, ensures production of natural antibodies expressing evolutionarily important specificities that are required for the initial defense against invading pathogens. Positive selection for reactivity with self antigens could also result in the production of detrimental anti-self antibodies. However, B-1 cells have evolved a unique response pattern that minimizes the risk of autoimmunity. Although these cells respond rapidly and strongly to host-derived innate signals, such as cytokines, and to pathogen-encoded signals, such as lipopolysaccharide and phosphorylcholine, they respond very poorly to receptor-mediated activation. In addition, they rarely enter germinal centers and undergo affinity maturation. Thus, their potential for producing high-affinity antibodies with harmful anti-self specificity is highly restricted. The positive selection of B-1 cells occurs during the neonatal period, during which the long-lived self-renewing B-1 population is constituted. Many of these cells (B-1a) express CD5, although a smaller subset (B-1b) does not express this surface marker. Importantly, B-1a cells should not be confused with short-lived anergic B-2 cells, which originate in the bone marrow in adults and initiate CD5 expression and programmed cell death following self-antigen recognition. In summary, we argue here that the mechanisms that enable natural antibody production by B-1 cells reflect the humoral immune system, which has evolved in layers whose distinct developmental mechanisms generate complementary repertoires that collectively operate to maximize flexibility in responses to invading pathogens. B-2 cells, present in what may be the most highly evolved layer(s), express a repertoire that is explicitly selected against self recognition and directed towards the generation of high-affinity antibody response to external antigenic stimuli. B-1 cells, whose repertoire is selected by recognition of self antigen, belong to what may be earlier layer(s) and inherently maintain production of evolutionarily important antibody specificities that respond to pathogen-related, rather then antigen-specific signals.

Since the distinction was made between immunoglobulin-bearing (B) lymphocytes that give rise to antibody forming cells and thymus derived, Thy-l-bearing (T) lymphocytes responsible for a host of other immune functions, substantial effort has been directed toward finding individual cell surface markers that subdivide these populations. In the mid-1970s, the Lyt-1, Lyt-2, and Lyt-3 antigens were shown (with the assays then available) to be represented exclusively on T cells (1, 2) and to identify functionally distinct T cell subpopulations. Lyt-1 appeared to be restricted to the helper-amplifier subset, and Lyt-2 and Lyt-3 defined the suppressor-cytotoxic subset (3-6).The development of monoclonal anti-Lyt reagents increased the sensitivity with which these antigens could be measured and significantly changed the status of the Lyt-1 (Ly-1) 1 marker: quantitative immunofluorescence studies with the fluorescenceactivated cell sorter (FACS) z revealed that all Thy-l-bearing cells have some Ly-1 (8); that lower levels of Ly-1 on cytotoxic-suppressor cells explains the previous failure to detect this antigen on the Lyt-2,3 subset with cytotoxic depletion assays; and that the frequency of Ly-1 + cells in normal spleen, in fact, is usually slightly greater than the frequency of Thy-1 + cells in the same organ (9).Recent studies (10, 11) demonstrating the existence of Ly-l-bearing B cells (Ly-1 B) indicate that such cells account for at least part of this excess. These findings, documented by FACS analyses on cell populations from normal animals, are consistent with a variety of previous observations: small numbers of Ly-1 + cells are present in B cell areas of stained tissue sections in normal spleen (8); certain mouse B cell lymphomas synthesize and coexpress Ly-1 and IgM (7); old NZB mice tend to have increased numbers of Ly-1 bearing cells (12) that we have now shown to be IgMpositive and Thy-1-negative (unpublished observations); human B cell chronic lymphocytic leukemias tend to carry IgM and Leu-1 (13), a human cell surface molecule whose properties are homologous to mouse Ly-1 (14). Furthermore, a subpopulation * This work was supported in part by grants GM-17367, HD-01287, and CA-04681 from the National Institutes of Health.:~ Fellow of the American Cancer Society. t Several years after its initial description as the first in a series of lymphocyte differentiation antigens, Ly-1 was renamed Lyt-1 to reflect its apparently exclusive expression on T cells. We return here to the original usage since this antigen was recently demonstrated on B cell tumors (7) and we now report its presence on a subpopulation of normal B ceils. No other cell surface antigen is presently called Ly-l.2 Abbreviations used m this paper: Bi, biotin; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; Fl, fluorescein; Ly-1 B, Ly-l-bearing B cell; PFC, plaque-forming cells; PI, propidium iodide; R1A, radioimmunoassay; TR, Texas red. 202J. Exp. MEn.