Recruitment of leukocytes from the blood is the key feature of inflammatory responses to tissue damage or infection. Prominent in this cascade is the rapid influx of granulocytes and monocytes that subsequently differentiate into inflammatory macrophages and/or dendritic cells (DC) (1, 2). This paradigm of classical ‘type-1′ inflammation is firmly grounded in processes elicited by microbial infection or necrotic cell death (2). Inflammation driven by helminth infection or allergy represents a distinct challenge to the immune system, and is characterized by the recruitment of cells that produce the cytokine IL-4, including eosinophils, basophils, and CD4+ Th2 cells (3, 4). Like classical inflammation, ‘type-2′ inflammation also results in the accumulation of large numbers of macrophages in the affected tissues, (3, 5-7), yet despite this IL-4 and Th2 responses are often regarded as ‘anti-inflammatory’.

To examine type 2 inflammatory processes we profiled cell recruitment during infection with a nematode that resides within ‘sterile’ tissues. The rodent filarial nematode Litomosoides sigmodontis induces strongly Th2-biased responses (fig. S1A), and drives alternative macrophage activation in the pleural cavity (Fig. 1A), after migrating there from the skin (Fig. 1B). For comparison, intra-thoracic injection of thioglycollate was used as a benchmark of the classical inflammatory cascade (9), because when injected, it induces recruitment of macrophages to the pleural cavity. Indeed, thioglycollate injection resulted in the rapid influx of neutrophils (Gr-1^high/Ly-6G^high) and Gr-1^{intermediate (int)}/Ly-6C^high monocytes, leading to accumulation of large numbers of F4/80⁺ macrophages by day 3 (Fig. 1C and fig. S1B). In contrast, L. sigmodontis infection triggered very little neutrophil or Gr-1^int/Ly-6C^high monocyte recruitment to the pleural cavity during the first 15 days after infection (Fig. 1D and fig. S1C), even though larvae arrive at this site between 3 and 6 days post-infection (Fig. 1B). Despite this, large numbers of F4/80⁺ macrophages gradually accumulated in the cavity after day 6 post-infection (Fig. 1D), accompanied by their conversion to an alternatively activated phenotype (Fig. 1A). There was also a striking difference in surface phenotype of the macrophage populations elicited by nematode infection compared to classical inflammation: those elicited by infection expressed levels of F4/80 similar to resident F4/80^high macrophages from naïve tissues (fig. S1C), whereas those induced by thioglycollate expressed low F4/80 levels (fig. S1B), which is characteristic of macrophages recruited to the serous cavities under inflammatory conditions (10).

These data raised the possibility that the large increase in macrophages seen during infection was the result of expansion of the resident population rather than blood cell recruitment. Whilst terminal differentiation of most mammalian cells typically results in reduced proliferative capacity, there is evidence that cells of the mononuclear phagocyte system can self-replicate in the periphery during development (14) or to maintain the tissue resident population (15, 16), but not as a classical inflammatory mechanism. Accordingly, on average 17% of resident pleural macrophages in naïve mice were positive for Ki67, a marker of proliferation expressed throughout cell-cycle, and 1% were in S-phase, as measured by a 3hr pulse of BrdU (Fig. 2A&B and fig. S3A). In contrast, less than 2% of thioglycollate-elicited macrophages expressed Ki67 and only 0.2% were in S-phase (Fig. 2A). Remarkably, pleural macrophages isolated ten days after infection with L. sigmodontis exhibited a substantial increase in the frequency of dividing cells. On average 38% of cells in the pleural cavity were positive for Ki67 and 6% were in S-phase (Fig. 2B). BrdU⁺ macrophages seen in the pleural cavity were not recruited from the blood because no blood monocytes incorporated BrdU in this 3 hour period (fig. S3B).

IL-4 is important for macrophage accumulation during numerous helminth infections (5, 6). We thus infected IL-4-deficient mice with L. sigmodontis to test the requirement for IL-4 in proliferative expansion of tissue macrophages. Significantly fewer macrophages from IL-4-deficient mice were capable of proliferating, as determined by BrdU incorporation, leading to a failure of these cells to accumulate in the pleural cavity (Fig. 2C). As expected, macrophages that were present in IL-4-deficient mice were also unable to M2 activate, whereas worm burdens were indistinguishable between strains at this early stage of infection, as previously described (18). To demonstrate that IL-4 drives macrophage proliferation in vivo, mice were injected via the intraperitoneal (i.p.) route with recombinant IL-4 complexed to IL-4 antibody (IL-4c), which increases the bioactive half-life of the cytokine (19). Delivery of IL-4c to naïve mice led to an elevation in peritoneal cellularity over a 4 day period that was due primarily to increased macrophage numbers, all of which exhibited an alternative activation phenotype (Fig. 3A). IL-4-elicited macrophages expressed very high levels of F4/80 (mean fluorescence intensity (MFI) of 7300 ± 680 versus 4900 ± 380 in PBS-treated controls; mean ± SEM) and neither inflammatory monocytes nor neutrophils were recruited (fig. S5), consistent with a process distinct from classical inflammation (2, 10). Critically, the rate of macrophage proliferation was increased compared to mice receiving PBS alone (Fig. 3A), whereas no proliferation or accumulation was observed in IL-4Rα-deficient mice (fig. S6). Thus, delivery of exogenous IL-4 was able to mimic the induction of proliferation and accumulation of macrophages observed during type-2 infection.

To establish definitively whether IL-4-driven accumulation of tissue macrophages is due to enhanced turnover of the resident population, we utilized a modified technique for creating bone marrow chimeric animals (20). Engraftment of bone marrow cells into lethally irradiated mice results in the replacement of most resident peritoneal macrophages by cells from transplanted bone marrow (16, 20). In the modified protocol, the upper body of Cd45.1 congenic mice was shielded during irradiation to protect resident pleural macrophages from harmful effects of direct exposure while allowing engraftment of donor CD45.2⁺ bone marrow. Mice were reconstituted for 8 weeks before i.p. injection of IL-4c or PBS, or intra-thoracic injection of thioglycollate. As expected, partial shielding led to a mixed chimerism in peripheral blood monocytes (Fig. 3D) and granulocytes (fig. S7) in all groups of mice, with approximately 25% to 30% of donor origin. In contrast, only 3% of pleural macrophages were of donor origin in PBS-injected mice (Fig. 3D), which demonstrated that little replacement of these cells occurred from the bone-marrow under steady-state conditions. Despite a 3-fold increase in cell number, the same low-level chimerism (approximately 3% donor-derived cells) was observed in pleural cavity macrophages of mice treated with IL-4c (Fig. 3D). Identical data were obtained for pleural macrophages from L. sigmodontis-infected chimeric mice (fig. S8). In contrast, macrophages elicited by injection of thioglycollate exhibited a high frequency of chimerism indistinguishable from that observed in blood monocytes and consistent with their differentiation from newly recruited cells of bone-marrow origin (Fig. 3D). These data reveal that IL-4 and filarial nematode infection elicit macrophages by expansion of the tissue resident population, independent of recruitment of bone marrow-derived precursor cells.

The obvious benefit of classical inflammation is the rapidity with which large numbers of macrophages can accumulate in the tissues but the maintenance of a circulating precursor pool throughout the body is energetically costly. In contrast, the slower mechanism of local expansion in type-2 inflammation does not require enhanced bone marrow activity (Fig. 3D) and may deliberately avoid inflammatory cell recruitment and the associated potential for tissue damage. Indeed, IL-4 can actively block granulocyte recruitment during delayed-type hypersensitivity (25) and instruct macrophages to down-regulate production of pro-inflammatory chemotactic factors (26). This would be of particular benefit during wound healing, for example, where type 2-activated macrophages accelerate repair (22, 27, 28) and sustained classical inflammation results in a failure to heal (29, 30). Thus, we propose that proliferation at site is an alternative mechanism of inflammation that allows macrophages to accumulate in sufficient numbers to perform critical functions such as parasite sequestration or wound repair in the absence of potentially damaging cell recruitment. This has broad therapeutic implications in settings where Th2-driven macrophages have key positive or negative function, such as in wound healing, autoimmunity or the tumor micro-environment.